INTRODUCTION:

Dengue virus (DENV) is a single-stranded positive-sense RNA virus. It is transmitted by infected female Aedes aegypti and Aedes albopictus mosquitoes. DENV was identified in Japan by Susumu Hotta and Ren Kimura during the period of Second World War¹They injected the serum sample from dengue suspected US soldiers into the suckling mouse at Kolkata in 1944 and isolated the virus ^2.

There are four serotypes of dengue virus viz DENV-1, 2, 3 and DENV-4 (family Flaviviridae, genus Flavivirus). Worldwide about 100 million cases were reported suffering from dengue fever and 500,000 cases of dengue hemorrhagic fever (DHF) and approximately 18,000 deaths have been reported every year ^3.

DENV-2 serotype is known to cause severe dengue in comparison to other serotypes. The genome of dengue virus is approximately 11 kb in length and contains one open reading frame that encodes three structural components (capsid, pre-membrane, glycoprotein envelope) and seven different non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) ^{4, 5.}In India, National Vector Borne Diseases Control Programme, Ministry of Health and Family Welfare Govt. of India reported 507454 dengue cases and 934 deaths across India from 2015 to 2018 (http://nvbdcp.gov.in/index4.php?lang=1andlevel=0andlinkid=431andlid=3715). The symptoms of dengue infection include high-grade fever, severe joint pain, muscle pain, eye pain, body rash, frontal headache, nausea, vomiting, cough, sore throat and retro-orbital pain. Symptoms usually appear 4-6 days after dengue infection and last for up to 10 days. Secondary infection is more severe than primary infection due to antibody-dependent enhancement ^6.Majority of dengue cases are self-limiting but few cases are severe in the form of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The first outbreak of DHF/ DSS occurred in Delhi, India, from August through November in 1996 ^{7, 8.} Unfortunately, though a vaccine has recently become available for dengue but there have been safety concern with its wider use ^9.

Many medicinal plants and their metabolites are widely used to treat a variety of viral diseases like dengue, influenza, herpes, and chikungunya in many parts of the world^10-12.Therefore, we focussed on the extracts of medicinal plants which are maybe more effective, safer and less toxic than synthetic drugs. Supercritical fluid extraction (SFE) is a non-conventional method, which is more rapid and accurate for obtaining high yields and more purified compounds. Hence, there is a robust obligation to investigate and develop a novel anti-dengue agent which will help to reduce viral replication in dengue patients and control dengue outbreaks. Molecular docking is used for virtual screening for drug development on the basis of drug-protein/DNA interaction to form a stable complex with high efficiency and specificity ^13.NS1 is one of the important factors in replication and pathogenic effect of DENV. NS1 is a glycoprotein which is involved in its RNA replication and can be a powerful target for the screening of drugs against dengue virus. The present study investigates the anti-dengue effects of supercritical extracts of Andrographis paniculata, Berberis vulgaris, Carica papaya, Euphorbia hirta, Phyllanthus niruri and Tinospora cordifolia against the dengue-2 virus in the C6/36 cell line and in silico methods.

MATERIALS AND METHODS:

Chemicals and reagents:

The chemicals and reagents were used in the study include 3-(4, 5-dimethylthiazol-2-yl) -2, 5- diphenyltetrazolium bromide (MTT, Hi-Media, batch no.0000263610), phosphate buffer saline (PBS, Hi-Media, batch no. 0000313379), streptomycin sulfate (100μg/ml, Hi-Media, batch no. 0000187551) and penicillin (100 U/ml, batch no. BCBN 3112V) antibiotic was purchased from Sigma-Aldrich, USA. Chemical used in cell culture including minimum essential medium (MEM, batch no.0000319279) and trypsin (batch no. 0000285329) were purchased from Hi-Media Laboratories (Mumbai, India). Fetal calf serum (FCS) was purchased from Gibco (NV, USA, batch no.1584260). Chemical including, buffer, enzymes, dNTPs, dengue specific primer, and probes were commercially available in Geno-Sen’s Dengue S1-S4 PCR kit used in the study.

Plant material collection:

Six medicinal plants were selected for the present study on the basis of their ethnobotanical uses. Out of these six plants, five were collected from the Herbal Garden of M. D. University, Rohtak, India, and one plant was collected from Shimla (Himachal Pradesh). The details of collected plants from different sites with voucher number are given in (Table 1). Identification of each plant was done on the basis of taxonomic keys and comparing with available herbarium data at M.D University, Rohtak India. The plant parts were washed with tap water to remove the dust and again washed with distilled water after that shade dried at room temperature and pulverized.

Preparation and standardization of medicinal plants extract using supercritical fluid extraction (SFE) machine:

Extracts of medicinal plants were prepared by using Supercritical Fluid Extraction Machine (Applied Separation Inc. U.S.). Ten grams of the plant powder was loaded into the stainless-steel extraction cell of the machine. The method of extraction for each plant was standardized and optimized at different temperature and pressure. The solvent (CO₂) flow rates were varied from 1.6 ml/min with static-dynamic mode (1- hour static and 40 minutes dynamic mode). The prepared plant extracts were lyophilized in aqueous (Hyper Cool HC3110, Hanil Scientific Inc.). The dried extracts were weighed and stored at 4 °C for further use. The percentage yields of plant extracts were calculated as follows-

Weight of the extract obtained

% age yield = ––––––––––––––––––––––––––– × 100

Weight of dried material

Preparation of plant extracts stock for cytotoxicity and antiviral assays:

The plant extract stock solution was prepared on the basis of the solubility of the plant extracts in the minimum essential medium (MEM). The stocks of A. paniculata and E. hirta were prepared by dissolving 1000 µg extract/ml of MEM medium and the solution of extracts was diluted to varying concentration from 500 µg/ml to 3.9 µg/ml. P. niruri stock was prepared by using 2000 µg extract/ml of medium in concentrations ranging from 1000 µg/ml to 15.62µg/ml. B. vulgaris, C. papaya and T. cordifolia stocks were prepared by dissolving 3000 µg extract/ml of the medium at concentrations from 1500 to 23.43µg/ml. The dissolved extracts were filtered using a 0.22µm syringe filter (MILLEX® GV). Extracted stocks were preserved at -20ᵒC for further use.

Determination of cell viability by MTT assays:

The maximum non-toxic dose (MNTD) of plant extracts was determined by using the C6/36 cells (1x 10⁴cells/well) in 96 well plates (Nunc, Thermo Fisher Scientific, USA). The plate was incubated overnight at 28°C in a CO₂ incubator. The culture medium was removed after reaching the 80% cell confluent of C6/36 cell lines. The cells were treated with different concentrations of plant extracts stock in triplicates. The last well of 96 plates contained 100 µl of the growth medium with cell only (negative control). After incubation of 96h, the medium was discarded and replaced with 10μL of MTT salt solution (5mg/1ml in PBS, as stock) and incubated for 3 hours at 28°C in 5% CO₂ incubator. After that, the solution of MTT was discarded without disturbing the cells. Then, 100μl of DMSO was added into each well to stop the reaction. The plate was continuously shaken for 5 minutes till all the formazan crystals were dissolved. Subsequently, absorbance values were noted by using a microplate reader (Bio-Rad, USA) at 595 nm. The percentage of living cells was determined by comparison with cell control and calculates its absorbance value by using the following formula;

Absorbance treated cell Absorbance blank

Cell viability (%) = ––––––––––––––––––––––––––––––––––– × 100

Absorbance cells control Absorbance blank

Table 1 Details of selected medicinal plants

S. No	Plants Name	Voucher number	Family	Locally Name	Collected part	Collected area	Collection Time
1	Andrographis paniculata Burm f Wall ex Nees.	(NISCAIR/RHMD/ Consult/2019/3518-19)	Acanthaceae	Kalmegh	Whole plant	Rohtak	July
2	Berberis vulgaris L.	NISCAIR/RHMD/ Consult/2019/3555- 56-2	Berberidaceae	Barberry	Leaf	Shimla	November
3	Carica papaya L.	D.DUN 25636 (MDU 3201)	Caricaceae	Papita	Leaf	Rohtak	May
4	Euphorbia hirta L.	D.DUN147402 (MDU 6205)	Euphorbiaceae	Dhudhi	Leaf	Rohtak	July
5	Phyllanthus niruri L.	D.DUN 88371 (MDU 6209)	Phyllanthaceae	Bhumi amla	Leaf	Rohtak	July
6	Tinospora cordifolia Thunb Miers	D.DUN 15203 (MDU 302)	Menispermaceae	Giloy	Stem	Rohtak	October

Cell culture and virus isolation:

The C6/36 Aedes cell line (ATCC^® CRL-1660^TM) was maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 2mML- glutamine, penicillin (100 U/ml) and streptomycin (100μg/ml). Cultured cells were incubated at 28˚C in a humidified atmosphere, with 5% CO₂^14-15.The medium was changed twice a week. A total of 500 µl of an appropriate dilution of dengue-2 standard strain (Accession no. TR-1751, National Institute of Health, Nonthaburi, Thailand) was inoculated onto confluent of C6/36 cells in 25 cm²tissue culture flask. The inoculum was incubated at 28°C in the presence of 5% CO_2,with shaking every 5 minutes. After 45 minutes, the virus growth medium was added and the cells were further incubated and observed daily under an inverted (Magnus, India) microscope for the presence of any possible cytopathic effect (CPE). In the absence of CPE, cells were harvested on 9^th day. The quantification of dengue virus was analyzed in lysate by real-time RT-PCR by generating the calibration curve using the five known standards (10¹-10⁵copies/ml) of dengue serotypes. The assay was performed by using the ABI 7500 real-time PCR instrument and commercially available Geno-Sen’s Dengue 1-4 kit.

RNA extraction:

RNA was extracted from 140µl of cell culture lysate with the help of commercial RNA kit (QIAmp Viral RNA mini kit (Qiagen, Germany) following the manufacturer̕ s instructions. The extracted viral RNA was eluted in 50µl buffer and stored at -70ºC until use.

In vitro anti-dengue assay:

The anti-viral assay was performed in 96 well plates with monolayers of the C6/36 cell line. A 100µl viral suspension of DENV-2 (TR-1751) with 100 viral copies/well was treated with 100 µl of the non-toxic dose of selective plants extract into the 96-well plate. The antiviral assay was performed using cell-control (medium with cells only), a virus-control (cells with the virus only with back titration) and MNTD of plant extracts together with 100 copies/well of virus (MNTD of the extracts and the virus). After that, the plate was sealed with parafilm and incubated on ice for 45 minutes and shaken gently after every 10 minutes. After adsorption of inoculum, the medium was aspirated from the wells. Then 200µl virus growth medium was added without disturbing the cells layer. The experiments were carried out in triplicate. Further, the 96-well plate was incubated at 28°C in a CO₂incubator for 7 days. After proper incubation, the plate was frozen at -70°C and the lysates were harvested and RNA was extracted from each aliquot.

Quantitative anti-viral assay by RT-PCR:

Quantitative real-time PCR (RT- PCR) was performed to determine the anti-viral effect of selected plants extracts on DENV-2 based on TaqMan chemistry probe by using the commercially available Geno-Sen’s Dengue S1-S4 PCR kit. The kit contains a specific master mix (buffer, enzymes, dNTPs, dengue specific primer, and probes) for specific amplification and quantification of dengue viruses. Before starting, all the PCR reagents were thawed and mixed properly. The master mix was prepared following the kit manufacturer’s instructions. After that, the desired number of PCR tubes were prepared by adding 10 µl master mix and 15 µl of extracted RNA to each lysate tube along with 15µl of the standards (Dengue 1-4, S 1-5) as a positive control and 15µl of water (PCR grade) as a negative control. Then, all the reagents in the PCR tubes were mixed properly by pipetting up and down. The PCR tubes were closed and transferred into the ABI 7500 real-time. The reaction conditions of PCR were as follows: reverse transcription at 50°C for 15 min, denaturation at 95ºC for 10 min, as followed by 45 cycles of denaturation at 95°C for 15sec, annealing at 55°C for 30 sec and a final extension step at 72°C for 15 sec. The fluorescence emission data were collected during the annealing step.

Data Analysis:

The percentages of cell viability of supercritical plants extract concentrations and anti-dengue data were analyzed by Microsoft Excel 2007 with the help of Tukey's test (each treatment mean value different from each other and compared to control). Samples were assayed in triplicates. The results were expressed as mean ± standard deviations. The cell viability of each plant was calculated as-

Absorbance treated cell Absorbance blank

Cell viability (%) = ––––––––––––––––––––––––––––––––––– × 100

Absorbance cells control Absorbance blank

Preparation of ligand and protein structure:

The dengue virus has a highly conserved NS1 a non-structural glycoprotein. The 3D structures of plant ligand i.e andrographolide (PubChem IDs: 5318517) was downloaded from PubChem. The 3D structures of dengue viral non-structural proteins NS1 (PDB IDs: 4O6B) was downloaded from PDB (protein data bank). The prosthetic groups were removed and protein structures were minimized using chimera. Auto dock software (V4.2.6) was used for docking analysis between dengue NS1 protein and selected ligand andrographolide. The ligand was prepared by managing total torsions available making ligand a bit flexible molecule. The search algorithm used for docking was a Lamarckian Genetic Algorithm (4.2). Docking complex was saved using MGL tool and interaction was visualized by Lig Plot plus^16.

RESULTS AND DISCUSSION:

Optimization of supercritical condition for extraction:

The supercritical extraction conditions for the plants were optimized and the results are shown in Table 2. A temperature of 60ºC at 20Mpa pressure was optimized for B.vulgaris, C. papaya and P. niruri plants on the SFE extractor machine while 40ºC at 15 Mpa pressure was optimized for E. hirta, A. paniculata and T. cordifolia, respectively. The highest yield of supercritical extracts was obtained from the P. niruri plant (4.8%) while the lowest yield (0.9%) was obtained from the T. cordifolia plant. A. paniculata has some secondary metabolites such as diterpenoids, diterpene, flavonoid and lactones. Andrographolide is a diterpene lactone a major compound found from A.paniculata. In present study demonstrated that the A. paniculata supercritical extract showed complete inhibition at 40ºC temperature and 15Mpa pressure. In previous literature suggested that the andrographolide was obtained at 40ºC temperature and 10Mpa pressure¹⁷by SFE, nearby present study. Supercritical fluid extraction (SFE) is a new analytical technique which is safe, reliable and time-saving, with giving higher yield in minimum time and better-purified compound at different temperature and pressure compared to conventional methods.

Figure 1 Maximum non-toxic of plants (B and D plants denoted as 46.87µg/ml, MNTD value and plants A, C and E MNTD 31.25µg/ml, plant F denoted 23.43 µg/ml, respectively on C6/36 cells lines by MTT assay

Table 3 Quantitative real-time PCR analysis of selected medicinal plants SFE extracts comparison with virus control copy number

Plants	MNTD dose (μg/ml)	RNA copy number^aMean (copies/ml)	Anti-dengue activity^b	Anti dengue activity Percentage ^C(%)
Andrographis paniculata Nees	31.25	not detected	+	Complete inhibition
Berberis vulgaris L	46.87	1.85 × 10⁶	˗	-5.06
Carica papaya L	46.87	1.15× 10⁶	˗	34.71
Euphorbia hirta L	31.25	1.25× 10⁶	˗	28.70
Phyllanthus niruri L	31.25	2.99× 10⁶	˗	-70.0
Tinospora cordifolia Thunb	23.43	2.92 × 10⁵	˗	83.44
Virus control (100 copies/ml)	-	1.76 ×10⁶	˗	Virus control

^a Plant extracts maximum non-toxic dose with virus-infected cells (MNTD values with virus/100 copies/ml).

^b Plants extracts with anti-dengue activity indicated by (+) and with no effect on dengue virus by (-),

^C The percentage of reduction of the dengue virus RNA copy number was compared with the virus control (1.76 x 10⁶copies/ml) after 7 days post-infection.

In previous studies, Rothan et al²¹ have demonstrated that methanol and ethanol extracts of Vernonia cinerea and Tridax procumbers show potent inhibitory activity against DENV-2. Lee et al²² have reported the anti-DENV-2 properties of Phyllanthus species^. Sharma et al ²³ have demonstrated that silver nanoparticles of Andrographis paniculata, Phyllanthus niruri and Tinospora cordifolia showed potent inhibitory activity against the chikungunya virus^. Andrographolide, a diterpene²⁴is the chief secondary compound derived from A. paniculata which possesses inhibitory activity against viruses^25-26 A. paniculata has some secondary metabolites such as diterpenoids, diterpene, flavonoid and lactones. Andrographolide has been reported to have activity against several viruses like hepatitis B virus, herpes simplex virus, influenza virus, hepatitis C virus, chikungunya virus, dengue virus 2 and 4 serotypes^27-28.

Molecular docking with dengue non-structural proteins (NS1):

The molecular study revealed that andrographolide showed 4 interactions (H-bonding) against protein NS1 with the binding energy of -7.30 Kcal/mol and residues: Arg 257, Gly 259 and Tyr 260 having an H-bond distance 2.70Å, 3.20Å, 2.93Å and 2.98Å, respectively. Figure 3, showed the interaction between protein and ligand in ligplot where andrographolide shows maximum binding (-7.30 Kcal/mol) with NS1 dengue protein. The inhibition constant (Ki) value is 4.47µM and Vdw and ele interaction are combined within total binding energy. In the present study, andrographolide total binding energy is -7.30 Kcal/mol with -0.24 Kcal/mol electrostatic energy and -9.15Kcal/mol Vander Waals (Vdw) with other near field interactions and in other studies it was -4.527Kcal/mol with andrographolide²⁹. Dengue is still a challenge for most countries of the world and has become a global health problem. There are no commercial anti-viral drugs available in the market against it. Medicinal plant extracts and derivatives have a capacity to fight against many diseases due to their therapeutic properties^30-32. Human health is extremely affected by several viral diseases like Chikungunya virus, Herpes Simplex Viruses, Nipah Virus³³, Zika Virus²³, Dengue virus³⁴ and now COVID-19^35.

In previous literature, the andrographolide is a major compound present in A. paniculata which possess many antiviral activities. Drugs such as aspirin, colchicines, digoxin, morphine, quinine, quinidine, artemisinine, taxol, tubocurarine, ephedrine, vincristine, and vinblastine have been derived from various medicinal plants are used in the treatment of different diseases. The numbers of medicinal plants have been reported for therapeutic purpose against different types of diseases^36-42. So, medicinal plants are an important source for drugs designing. Therefore, research on medicinal plants extracts and their bioactive compounds which could be safer and cheaper than synthetic drugs is gaining importance ⁴³. Many other studies have demonstrated the value of medicinal plants against dengue serotypes^44-47.

Figure 3: Ligplot showing the amino acids involved in interactions with andrographolide

CONCLUSIONS:

The inhibition profile of six medicinal plant extracts for dengue-2 showed that A. paniculata and T. cordifolia extracts had significant anti-dengue activity as compared to others. As anti-dengue activity is associated with A. paniculata further research is required to focus on specific metabolite isolation and its the mechanism of action. Molecular docking revealed that the andrographolide of A. paniculata binding to the dengue NS1 proteins which inhibit dengue replication with higher binding energy. Hence, it is concluded that plant extracts have the potential to develop newer anti-dengue drugs with unique drug targets.

ACKNOWLEDGMENTS:

The research work was financially supported by UGC under UGC-SAP program (F.3-20/2012, SAP-II). Sulochana Kaushik acknowledges the financial support for the award of University Research Scholarship by Maharshi Dayanand University, Rohtak.

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Received on 12.08.2020 Modified on 16.11.2020

Research J. Pharm. and Tech 2021; 14(11):5895-5902.

DOI: 10.52711/0974-360X.2021.01025

S. no.	Plants name	Plant sample	Temperature °C	Pressure Bar	Yields
1	Andrographis paniculata Nees	10gm	40	150	1.8%
2	Berberis vulgaris L	10gm	60	200	3.5%
3	Carica papaya L	10gm	60	200	2.5 %
4	Euphorbia hirta L	10gm	40	150	1.3%
5	Phyllanthus niruri L	10gm	60	200	4.8%
6	Tinospora cordifolia Thunb	10gm	40	150	0.9%