Pakistan J. Zool., vol. 51(1), pp 395-398, 2019. DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.1.

SC11

Short Communication
Study of Antiviral Potential of Cholistani
Plants against New Castle Disease Virus
Mirza Imran Shahzad1, Hina Ashraf2,*, Muhammad Arshad3, Sabeeha Parveen4,
Amna Aslam4, Nargis Naz4, Zahid Kamran1, Sumbul Gohar Khalid5, Sajid Hameed1,
Muhammad Ashfaq6 and Muhammad Mukhtar7
1
University College of Veterinary and Animal Sciences, The Islamia University, Bahwalpur
2
Department of Botany, Govt. Sadiq College Women University, Bahawalpur
3
Department of Basic Sciences, College of Veterinary and Animal Sciences, Jhang
4
Department of Botany, The Islamia University, Bahawalpur Article Information
5
Department of Bioinformatics and Biotechnology, Islamic International University Received 28 September 2017
Revised 12 January 2018
Islamabad Accepted 28 March 2018
6
Department of Chemistry, The Islamia University of Bahawalpur Available online 11 January 2019
7
Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Malaysia
Authors’ Contributions
MIS, NN and MM supervised the
ABSTRACT work. HA, SP and AA conducted the
research. MIS designed. MA, SKG
Present study is based on evaluation of antiviral potential of methanolic extracts of eleven selected and SH facilitated in development of
Cholistani plants against New Castle Disease Virus (NDV)-LaSota strain. All of these plants were antiviral assays. ZK and MA provided
reported for their different pharmacological activities but their antiviral potentials were not known administrative and financial support
before. The methanolic extracts were made and concentrated by rotary evaporator and finally dissolved to this research.
in distilled water before taking their antiviral trials in 7-11 days old chicken embryonated eggs. The
viral loads were determined through heamagglutination (HA) test. The methanolic extract of each plant Key words
NDV, Antiviral activity, Cholistan, In
was found effective against NDV but in varying order. The active extracts were further used in different
Ovo assay.
concentrations and IC50 of each extract was calculated. The extract of Achyranthes aspera was found most
effective with IC50 3.125μg and the extract of Oxystelma esculentum, was found least effective with IC50
60μg in the series. The current study has established the fact that Cholistani plants are rich source of anti-
NDV agents and can be used in purified or in crude form.

E xploiting plants for medicinal purposes is the biggest

example of human’s utilization of nature. Although
market is now flooded with no. of antimicrobial agents
Other plant based principal antiviral agents include pro-
teins, polypeptides, peptides, furyl, sulphides, flavonoids,
poly phenolics, terpenoids, coumarins and saponins (Cow-
but demand of new drugs especially antiviral drugs is an 1999). These plant based compounds are not only safe
continuously rising. Extracts, powders, herbal teas, oil to use, easy and cheap to produce but also found more ef-
based emulsions of plants are already in use to treat viral fective as compared to synthetic compounds. The studies
diseases from hundreds of years but demand for new have proved that plant based products work in more syner-
compounds is increasing (Vijanyan et al., 2004). Higher gistic way and provide less chance to microbes to develop
plants are rich source of antiviral drugs (Jassim and Naji resistance against them (Solanki, 2010).
2003; Aslam et al., 2016; Shahzad et al., 2017). Recently Newcastle disease (ND) or commonly called as Rani
a compound Nordihydroguaiaretic acid (NDGA) was Kheit is a highly infectious viral disease which affects
isolated from leaves of Larrea divaricate and found very poultry of all ages (Shahzad et al., 2011). ND outbreaks
effective against Junin virus (Konigheim et al., 2005). were on continuous rise during the period of 2011- 2012 in
Similar, compound was isolated from other species of genus country. Most of the young commercial broiler flocks were
Larrea and successfully tested against Herpes Simplex damaged. There is no known treatment of ND in market
Virus (HSV) type I and II. These NDGA compounds except extensive use of vaccine (Kitazato et al., 2007). By
are lignin in nature and they have inhibitory effect on keeping in view the devastating effects of ND on poultry
transcription process of HIV viruses (Gnabre et al., 1995). industry and natural resourcefulness of Cholistani plants,
the current study was designed to evaluate their antiviral
* Corresponding author: [email protected] potentials against NDV and provide cost effective remedy
0030-9923/2019/0001-0395 $ 9.00/0 from local resources.
Copyright 2019 Zoological Society of Pakistan
396 M.I. Shahzad et al.

Materials and methods Results and Discussion

Eleven fresh plants including Achyranthes The desired HA titer i.e. 2048 of NDV was achieved
aspera,Haloxylon recurvum,Haloxylon salicornicum, after 6th passage.
Oxystelma esculentum,Octhocloa compressa,Neurada In initial experiments, when 100 mg dose of plant
procumbens, Panicum antidotale, Salsola baryosma, extracts were tested against NDV eight out of eleven
Suaeda fruticosa, Sporobolus icolados and Solanum extracts were found very effective and kept HA titers at
surattense were collected from herbarium and surroundings 0. The remaining three plant extracts were also effective
of Cholistan Institute of Desert Studies, Baghdad ul Jadeed but their HA titers were 8 (Table I). In later experiments,
Campus, The Islamia University of Bahawalpur. The different concentrations of plant extracts were were made
plants were identified and authenticated by taxonomists and tested against NDV and IC50 value of each extract
at Department of Botany, The Islamia University of was calculated. The methanolic extracts of A. aspera
Bahawalpur and their vouchers were saved. New Castle and H. salicornicum were best among all and their IC50
disease virus (NDV) was obtained from depository of values were 3.125 and 6.25 mg, respectively. Among
Biochemistry and Molecular Biology Lab, University other plants the extracts of S. surattense, S. fruticosa
College of Veterinary and Animal Sciences, The Islamia O. compressa and H. recurvum have shown 100% anti-
University of Bahawalpur and specific pathogen free NDV activity in their higher concentrations including
(SPS) 9-11 days chicken embryonated (CE) eggs were 100 and 75, respectively but these plants lost the potential
obtained from Govt Hatchery, Model town A, Bahawalpur. gradually in lower concentrations (Table I). The extracts
The NDV was propagated in 9-11 days old CE eggs via of plants N. procumbens and O. esculentum were next
chorioallantoic route (Sulaiman et al., 2011). in order and they have controlled NDV very effectively
For preparation of methanol extracts, the whole plants only in higher concentrations (100 mg) but later they lost
were collected and dried under shade at room temperature their potential gradually. The potency of these extracts
(RT) for 10 days. The dried plants were grinded by lectric has direct relationship with concentration of extracts and
grinder and kept in airtight containers at RT. Methanol their antiviral activities. The antiviral potentials of plant
extracts were made by dissolving 10 g powder of each plant extracts of P. antidotale, S. baryosmaand S. icolados, were
in 200 mL methanol. The suspensions were kept in airtight least effective in antiviral category and none of them has
containers for 96 h at constant shaking. Later, the samples reduced HA titer at 0 through any concentration (Table I).
were subjected to rotary evaporator and concentrates All plant extracts were found effective against NDV
were rinsed with methanol and chloroform mixture and but in varying order. Initially, all plant extracts were tested
allow them to re-evaporate. Finally the precipitates were at higher concentrations i.e. 100 mg all of them were
weighed and dissolved in 10 mL distilled water, vortexed, found effective against NDV. It was worth mentioning
filter sterilized and stored at -20 °C (Joshi and Kaur, 2013. that all plant extracts were found nontoxic at 100 mg
For viral inoculations, 9-11 days old CE eggs were and all embryos were found alive after inochoulations
candled before inoculations. The broader ends of eggs and similarly, no change in shape of RBCs, rupturing of
were swabbed with 70% alcohol and drilled manually blood vessels and hemolysis was seen by these extracts.
with sterile needle. The inoculum was injected and hole The extracts of A. aspera and H. Salicornicum plants were
was sealed with molten wax. The inoculated eggs were the most potent and rich source of anti-NDV agents. These
incubated at 37°C for 48 h. Later the eggs were harvested extracts were equally effective at 25 mg concentration as
and HA test was performed (Wang et al., 2008). they were effective at 100 mg concentrations. The extracts
Standard HA test was optimized to quantify the titer of rest of plants were also found effective but in varying
of ND virus. order. Even the lowest ranked extract of S. baryosma, was
For in ovo antiviral assay, the filter sterilized plant active against NDV at 100 mg concentration. These results
extracts were mixed with equal vol. of viral inoculums and indicate that Cholistani plants are naturally rich source of
injected in 9-11 days old CE eggs. Different concentrations anti-NDV agents. There is no known drug against NDV
of plant extracts (25-100 mg) were tested against NDV. except few herbal products (Mabiki et al., 2013). Similar
Autoclave distilled water was used as negative control studies were done by Bedoya et al. (2001) on plants from
and NDV as virus control. HA titers were determined after Iberian Peninsula and screened various medicinal plants of
each experiment and change in HA titer as compared to this region for their anti HIV activity on HIV-infected cells.
virus control was noted (Table I). IC50 of each extract was Wang and Lu (1995) has reported antiviral activity from
calculated by serial dilution method. extracts of Geranium carolinianum L. In another report,
its aqueous extract was found effective against HBV and
help in improving the clinical data of patients (Zhu and
397 Anti-NDV Activity from Cholistani Plants 397

Ren, 1995). In recent studies done by Gescher et al. (2011) The antiviral research study against AIV by Ehrhardt
the aqueous extract of Rhododendron ferrugineum was et al. (2007) provides the basis for further research on
positive against Herpese Simplex Virus type 1 (HSV-1) plant based anti-adhesive compounds. The results of these
while negative against adenovirus. studies were in accordance of results of other researchers
like Gonzalez et al. (2012). They have reported a plant
Table I.- Anti-NDV activities from methanolic extracts based compound Fucoidan from C. Okamuranus, which
of Cholistani plants and their IC50. was found very active against NDV. Similarly antiviral
activities against Vaccinia, NDV and HBV were reported
Medicinal Concent. of HA titer IC50 Control
by Premnathan et al. (1992) by using methanol extracts of
plant extract (mg/0.1 after (mg/0.1 virus
mL H2O) challenge mL H2O) mangroves, seagrasses and seaweeds, of India’s southeast
S. surattense 25 8 6.25 1024 coastal region. Similarly, Usha and Sharma (2012) have
50 4 2048 tested aqueous, ethanol and methanol extracts of C. crista
75 0 1024 L. and found them effective against NDV. Similar results
100 0 2048 were found from fruit pulp and leaf extracts of Momordica
A. aspera 25 4 3.125 2048 balsamina against NDV (Chollom et al., 2012). Similar
50 0 1024 studies against other poultry viruses are also reported
75 0 2048 like Ahmed et al. (2014) has reported anti-IBDV activity
100 0 2048 from ethanol extracts of Glycyrrhiza glabra, Moringa
S. baryosma 25 512 50 1024 oleifera, Phyllanthus emblicus and Eugenia jambolana.
50 128 2048 Pant et al. (2012) have proved that hydro- alcoholic
75 32 2048
extract of Withania somnifera roots was rich in anti-
100 8 1024
IBDV compounds. Similarly, Park (2003) has reported
O. 25 512 60 2048
esculentum 50 256 2048 anti-AIV activity from methanol extracts of four different
75 64 1024 Korean plants. Ramzi et al. (2006) have reported antiviral
100 0 2048 activities against AIV and HSV-1 from their methanol
P. antidotale 25 32 12.5 1024 and hot aqueous extracts of Boswellia ameero, Boswellia
50 32 2048 elongata, Buxus. hildebrandtii, Cissus hamaderohensis,
75 16 2048 Dracaena cinnabari, Exacum affine, Cleome socotrana,
100 8 2048 Jatropha unicostata and Kalanchoe farinacea. Chen et al.
S. icolados 25 32 12.5 1024 (2014) have studied moderate anti infectious bronchitis
50 32 1024 virus (IBV) activity from ethanol extracts of Sambucus
75 8 2048 nigra fruit. Different researchers have suggested different
100 8 4096
mechanisms of antiviral activities from different extracts
O. 25 32 6.25 1024
of medicinal plants, some have anticipated the loss in
compressa 50 08 2048
75 0 1024
attachment of the virus to host cell surface especially
100 0 2048 at early stages of infection or some have suggested the
S. fruticosa 25 32 12.5 1024 inhibition of essential enzymes required for growth or
50 4 2048 multiplication of virus. Some have reported the protein
75 0 1024 or polypeptide nature of antiviral compounds (Bajpai and
100 0 2048 Chandra, 1990).
H. recurvum 25 256 35 2048
50 32 2048 Conclusions
75 0 2048 There is no known treatment of New Castle
100 0 2048 Disease except some herbal extracts. Cholistani plants
H. 25 32 6.25 2048
have highlighted huge antiviral potential to treat NDV.
salicornicum 50 0 2048
Although Cholistani plants were reported for number of
75 0 2048
100 0 2048
pharmacological activities but this is the first report of
N. 25 128 25 2048 their antiviral potential especially against NDV.
procumbens 50 64 2048
75 4 2048 Acknowledgements
100 0 2048 We acknowledge the services provided by Govt.
Poultry Farm, Model Town A Bahawalpur in terms of
398 M.I. Shahzad et al.

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