Sophora interrupta Bedd. root-derived flavonoids as prominent antiviral agents against Newcastle disease virus†

Cherukupalle Bhuvaneswar^ad, Aluru Rammohan*^be, Baki Vijaya Bhaskar^g, Pappithi Ramesh Babu^a, Gujjar Naveen^c, Duvvuru Gunasekar^b, Subbiah Madhuri*^c, Pallu Reddanna^f and Wudayagiri Rajendra*^a
aDepartment of Zoology, Sri Venkateswara University, Tirupati-517502, AP, India. E-mail: rajendraw2k@yahoo.co.in
^bNatural Products Division, Department of Chemistry, Sri Venkateswara University, Tirupati-517502, AP, India
^cNational Institute of Animal Biotechnology (NIAB), Hyderabad-500049, AP, India
^dDepartment of Microbiology, Sri Venkateswara University, Tirupati-517502, AP, India
^eDepartment of Organic and Biomolecular Chemistry, Ural Federal University, Yekaterinburg 620002, Russian Federation
^fSchool of Life Sciences, University of Hyderabad (UOH), Hyderabad-500046, Telangana, India
^gDepartment of Pathophysiology, Shantou University Medical College, Shantou, Guangdong, China-515031

First published on 10th September 2020

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1. Introduction

Among all the poultry diseases, influenza (HPAI) and Newcastle disease (ND) are the two foremost diseases that have caused economically devastating results in poultry farms, which in turn affects the human health due to malnutrition.^1,2 NDV can infect more than 250 bird species across the world.³ The onset of Newcastle disease is rapid and clinical signs involving respiratory, digestive, and/or nervous systems appear within 48 h. The severity of this disease (high morbidity and mortality) depends on the infecting strain and the bird's susceptibility.³ The currently used vaccines are not 100% protective and disease outbreaks have been recorded in vaccinated flocks. On the other hand, antiviral drugs are not ideal due to their side effects, expensive treatment procedures, and moreover, the fear of accumulation of residues in meat and/or eggs. For example, Ribavirin a well-known broad-spectrum antiviral drug against various RNA viruses^4–6 and approved for the treatment of respiratory syncytial virus (RSV) frequently results in lower respiratory tract infections in children. However, there are some concerns about its efficacy due to its potential toxic effects on the exposed individuals when administered via aerosol and the high price of the drug.^7,8 All these factors reiterate the necessity for the identification of novel antiviral drugs from natural sources.^9,10 Although a few plant extracts have shown good anti-NDV activity,¹¹ none of them are in commercial use. It may be due to insufficient work in the area of the active component(s) identification, which is accountable for the antiviral activities and their mechanism of action. Hence, it is essential to identify and characterize bioactive component(s) from natural sources that could display promising antiviral activities.

Plants are a rich source of new and novel natural compounds^12–14 for the development of active pharmaceutical ingredients. Sophora interrupta Bedd. belongs to the family Fabaceae, which is an endemic medicinal plant of Tirumala Tirupati hill ranges of Chittoor district, Andhra Pradesh, India.¹⁵ The genus Sophora is commonly used in Indian Ayurvedic medicine as well as traditional Chinese medicine as a remedy to cure several diseases and disorders.^16–20 Two flavonoids, anagyrine and sophoranol, were isolated from Sophora flavescens, which were found to show antiviral activity against RSV.²¹ Earlier reports on S. interrupta resulted in few flavonoids, isoflavonoids, coumarins, and other phenolic compounds.^22,23 Besides, our preliminary investigations have revealed that the root extracts of Sophora interrupta showed less oxidative stress and lowered the viral titer in NDV-infected chickens.²³ Even though few biomolecules have been isolated from Sophora interrupta Bedd., to the best of our knowledge, there is no antiviral report on this endemic plant (except for our preliminary investigations) and hence, it was considered for the current investigations. Therefore, the present work is intended to isolate and characterize the antiviral compounds from the methanolic root extract of Sophora interrupta Bedd. and to evaluate their antiviral activities in order to optimize the lead molecules against Newcastle disease viral infections.

2. Materials and methods

2.1. General experimental procedures

All chemicals and solvents were of analytical grade, were purchased from Merck, India, and used without further purification. HPLC analysis was carried out using a Shimadzu HPLC system equipped with a binary gradient system with a variable UV-Vis-detector (SPD-20A) comprising a Rheodyne injection valve with a loop size of 20 μL, LC-20AD pumps, and an integrator. Thin layer chromatography (TLC) was performed by using silica gel 60 F₂₅₄ pre-coated plates (Merck) and column chromatography was performed on 100–200 mesh size silica gel (0.08 mm thickness, Acme). Melting points were determined using a Kofler hot stage apparatus and are uncorrected. The ESI-TOF-MS spectra were recorded in the positive mode on an API Q-STAR PULSA I spectrometer of Applied Biosystems. UV spectra were measured in MeOH on a Shimadzu UV-1800 spectrophotometer. The IR spectra were recorded using a Bruker Alpha-Eco ATR-FTIR spectrophotometer, and the ¹H and ¹³C NMR spectra were recorded on a Bruker Avance spectrometer operating at 600 MHz for ¹H and 150 MHz for ¹³C, using DMSO-d₆ with TMS as the internal standard.

2.2. Collection of the plant material

The roots of Sophora interrupta Bedd. were collected from Kumaradhara Theertham of the Tirumala-Tirupati hill ranges, Chittoor district, AP, India. The plant species was identified and authenticated by Dr K. Madhava Chetty, Plant Taxonomist, Department of Botany, Sri Venkateswara University, Tirupati, India, wherein a voucher specimen (No. 721) was deposited.

2.3. Extraction of the plant material

Solvent extraction was carried out by the method described by Parekh and Chanda²⁴ with slight modifications. 500 g of shade-dried and powdered roots of Sophora interrupta was soaked in MeOH in the 1:5 ratio (w/v) in conical flasks plugged with aluminum foils. After proper soaking for 48 h, the solvent was collected and re-extracted thrice, and the supernatant solvents were pooled and filtered using Whatman No. 1 filter paper after treating with charcoal powder. The combined methanol extracts were concentrated under reduced pressure using a Büchi rotary evaporator. The solvent-free extract was stored at 4 °C in air tight bottles for further study.

2.4. Isolation of the flavonoids

The concentrated methanol extract (25 g) was refluxed with n-hexane to remove oils and fats, and the n-hexane insoluble portion was eluted with ethyl acetate. Further, the ethyl acetate soluble fraction (13 g) was separated over a silica gel (100–200 mesh) column using n-hexane–ethyl acetate step gradient solvent system. The n-hexane–ethyl acetate eluents, with concentrations in the ratio of 7:3 and 6:4, were employed for the concentration of the separated fractions, followed by crystallization, which yielded colorless prisms (SR-1, 43 mg) and a yellow crystalline solid (SR-2, 47 mg), respectively (Fig. 1).

2.5. Analyses of the root extract and isolates by HPLC

The methanol extract and the isolated compounds were qualitatively analyzed by High Performance-Liquid Chromatography (HPLC), according to the standard procedure.²⁵ Reverse phase chromatographic analysis was carried out in isocratic conditions using 0.4% methanol and acetic acid (80:20 v/v) as the mobile phase over a C-18 reverse phase column at 25 °C with a flow rate of 1 mL min⁻¹. The compounds were detected at 293 nm wavelength using a UV-Vis detector.

2.6. Cell lines

Chicken fibroblast (DF-1) cells were obtained from American Type Culture Cells (ATCC-CRL-12203), USA, and they were revived and sub-cultured in BSL-II laboratory. The DF-1 cells were cryopreserved in a liquid nitrogen tank (−196 °C) for further use. Dulbecco's Modified Eagle Medium (DMEM) was supplemented with 10% Fetal Bovine Serum (FBS), penicillin, and streptomycin were used for routine sub-culturing and for the in vitro experiments.

2.7. Cytotoxic assay

The isolated compounds SR-1 and SR-2 were tested for their cytotoxic effects on chicken embryo fibroblast cell line (DF-1) by the MTT [3-(4,5-dimethylthiazol-2-ol)-2,5-diphenyltetrazolium bromide] assay, according to the method described by Mosmann²⁶ with slight modifications. The DF-1 cells were seeded onto 96-well cell culture plates at a density of 1.5 × 10⁴ cells per well in DMEM supplemented with 10% FBS and incubated for 6–8 h to allow the cells to form a monolayer. The cells were incubated with increasing concentrations of isolated compounds (3–500 μg mL⁻¹) in triplicates and allowed to proliferate for 72 h at 37 °C in 5% CO₂. The culture medium was discarded and the cells were subsequently washed with phosphate-buffered saline (PBS) thrice. 20 μL of the MTT reagent at a concentration of 5 mg mL⁻¹ was added to each well for the development of formazan. Further, 100 μL of MTT-solubilizing solution was added to each well of the plates and the plates were incubated at 37 °C in 5% CO₂ for 1 h to solubilize the formazan crystals. The absorbance was measured at 570 nm using a multi-mode plate reader. The cytotoxicity of the compounds was expressed as CC₅₀ and Maximum Non-Toxic Concentration (MNTC), which is the safest concentration at which 100% cells are viable. Thus, the percentage of cell viability was calculated at this concentration.

2.8. Quantification of viral RNAs

2.9. Statistical analysis

All the experiments were carried out in triplicate and calculated using SPSS v16. The data was expressed as mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance (ANOVA), followed by student's “t” test and the findings were considered to be statistically significant if p-value is <0.05.

2.10. In silico studies

3. Results and discussion

3.1. Isolation and characterization of flavonoids

The methanol extract of the roots of Sophora interrupta was prepared using the standard protocol and concentrated under reduced pressure, which yielded a dark brown gummy solid. To testify the active compound profile of the methanol extract, it was subjected to Reversed-phase High Performance Liquid Chromatography (RP-HPLC) analysis and the crude extracts showed five major peaks with retention times at 2.14, 2.85, 4.92, 5.74, and 8.87 min (Fig. 2A). Further, the methanol extract was subjected to purification over a silica gel column, which yielded two compounds that were designated as SR-1 and SR-2. Thus, the successfully isolated purity of the two compounds was analysed by using HPLC with retention time of 5.0 and 8.8 min, and compared^25,32 with the crude extract profile (Fig. 2B and C). The chemical structures of the isolates (Fig. 1) were established by using UV, FT-IR, ¹H NMR, ¹³C NMR, and ESI-TOF- mass spectral analysis and were characterized as 3-hydroxy-8,9-methylenedioxy-pterocarpan (SR-1) and 2′,4′-dimethoxy-3′-(γ,γ-dimethylallyl)3,5,7-trihydroxy-isoflavanone (SR-2) as their spectral data were in good agreement with the literature values.^33,34

3.2. Cytotoxicity assay

Subsequently, the cytotoxicity profiles of SR-1, SR-2, and the standard drug Ribavirin were determined in chicken embryo fibroblast DF-1 cell lines by the MTT assay.²⁶ The maximum non-cytotoxic concentration (MNCC), at which no significant changes were detected in the cellular morphology of the DF-1 cells of SR-1 and SR-2, was found to be 62.5 and 40 μg mL⁻¹, respectively, while that of Ribavirin was 31.25 μg mL⁻¹. This suggested that both the compounds are comparatively less toxic than Ribavirin. Based on the results of the MTT assay, the 50% cytotoxic concentration (CC₅₀: the dose that inhibited the growth by 50% compared to the untreated cells) of SR-1 was determined as 600 μg mL⁻¹, while that for both SR-2 and Ribavirin was 400 μg mL⁻¹.

3.3. Viral plaque assay and plaque reduction assay

Further, the viral plaque assay was performed to evaluate the antiviral effect of the isolated compounds SR-1 and SR-2, and compared with that of the standard Ribavirin in DF-1 cells. The isolates and Ribavirin with different concentrations such as 10, 20, 30, and 40 μg mL⁻¹ were used before, after, and during the NDV infection in order to categorize the stage of the viral life cycle that was probably inhibited.^35,36 The percentage of plaque reduction was determined by calculating the reduction in the number of plaques during compound treatment and compared to the untreated NDV infected cells, which were defined as 100%. Concisely, DF-1 cells (1.5 × 10⁵ cells per well of a 24-well plate) were infected with 10-fold serial dilutions of NDV laboratory stock (2⁹ hemagglutination units), followed by overlaying with Dulbecco's Modified Eagle's Medium containing 0.8% methylcellulose without serum supplementation and incubated at 37 °C in 5% CO₂ incubator. After 4 days, the cells were fixed and stained with 1% crystal violet to visualize the viral plaques. At 10⁻⁵ μL dilution of the stock virus, 50 plaques were observed and this viral dilution was subsequently used as the standard viral infection dose for the entire study (Fig. 3).

To determine whether the test compounds inhibit virus adsorption, DF-1 cells were treated with the compounds for an hour prior to NDV infection. There was a significant reduction in the plaque numbers in the treated cells compared to the untreated controls. The inhibitory concentration (IC₅₀) of SR-1, SR-2, and Ribavirin was 10, 20, and 40 μg mL⁻¹, respectively. SR-1 and SR-2 exhibited strong antiviral activity by decreasing the viral plaques by 67% and 59%, respectively, at their maximum test dose, compared to only 32% viral plaque reduction by Ribavirin at MNCC (Fig. 4A). The results suggest that both SR-1 and SR-2 can significantly inhibit viral adsorption and thereby prevent NDV infection at a lesser dose (higher therapeutic index) compared to Ribavirin. In the second assay, the compounds were added to the NDV-infected cells 1 h post-infection. Both IC₅₀ and the therapeutic index (TI) values could not be determined at the test doses. A moderate reduction in the viral plaque number was observed compared to the previous experiment; SR-1 and SR-2 showed 38% and 35% of viral plaque reduction at their maximum test dose (40 μg mL⁻¹), respectively, compared to only 18% by Ribavirin at MNCC (Maximum Non-Cytotoxic Concentration of 31.25 μg mL⁻¹) (Fig. 4B). The results indicated that the compounds probably interfered with the viral replication and/or transcription, similar to Ribavirin,³⁷ and further reiterated that the compounds could be better than Ribavirin with respect to the inhibition of NDV infection in vitro.

In the third experiment, the compounds were mixed with the virus, incubated at 4 °C for 1 h, and then the mixture was added to the DF-1 cells. SR-1 and SR-2 reduced the viral plaques by 21% and 52%, respectively, at their maximum test dose compared to 34% by Ribavirin at MNCC (Fig. 4C). The IC₅₀ for SR-2 was found to be 30 μg mL⁻¹ but the IC₅₀ values could not be determined for SR-1 and Ribavirin at the tested doses. The treatment of the cells with a mixture of the test compounds and the virus could have led to the competition between the virus and the test compounds to bind to the host cell receptors, or the test compounds could have altered the structure of either the viral surface antigen or the host receptors, thereby reducing the binding efficiency of the virus to the host cell receptors. Also, it may conceivable that the compounds could inhibit the early stages of viral replication such as endocytosis and uncoating, as reported earlier.³² The MNCC, CC₅₀, IC₅₀, and TI values of SR-1, SR-2, and Ribavirin are summarized in Table 1.

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3.4. Quantification of viral RNAs

NDV carries a single stranded, non-segmented, and negative-sense RNA coding for six structural proteins, namely, nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin neuraminidase (HN), and large protein (L), as well as two non-structural (V and W) proteins.³⁸ NP encapsulates the viral genomic RNA (gRNA) and antigenomic complementary RNA (cRNA) to resist the host nucleases and to mediate viral RNA replication and transcription via RNA-dependent RNA polymerase.³⁹ To understand the mechanism(s) of anti-NDV activity, strand-specific quantitative PCR was performed to detect the NP region of NDV gRNA, cRNA, and mRNA in both the NDV-infected and compound-treated DF-1 cells as an indirect measurement of the viral replication and transcription.²⁷ In SR-1, SR-2, and Ribavirin-treated cells, the amounts of gRNA, cRNA, and mRNA were significantly reduced when compared with those in the viral control (Fig. 5). Tables S1 and S2† represent the primers and CT values, respectively. Fig. S1–S5† represent the supporting molecular data. The decline in the levels of all the three viral RNAs in comparison with their levels in the NDV-infected DF-1 cells (untreated virus control) clearly indicated that the compounds inhibit both NDV replication and transcription.

3.5. Docking studies and bioavailability

NDV has two district surface glycoproteins, namely, a hemagglutinin neuraminidase (HN) and a fusion (F) protein. Indeed, HN confers three significant roles: (i) it distinguishes the receptor with sialic acid sites on the cell surfaces; (ii) it enhances the F protein fusion activity and viral penetration ability on the cell surfaces; and (iii) it displays sialidase enzyme activity by removing the sialic acids from the progeny viral particles. However, the HN molecule aids in the designing of novel antiviral drugs using structure-based drug design approach against paramyxovirus diseases.

Therefore, in the present study, the docking experiments of isolated compounds maackiain (SR-1) and echinoisoflavonone (SR-2) were conducted using the HN protein. As a result, SR-2 showed greater binding affinity of −8.0 kcal mol⁻¹ than that of SR-1 with −7.6 kcal mol⁻¹. The functional groups present in compound SR-2 positioned within the sialic acid site of the HN protein, exhibiting foremost interactions with the hotspot residues such as the 2′ and 4′-dimethoxy residues, showed two bonds with Lys236 and Ser237, 3-OH bond with Tyr526, 5-OH group with Arg363, whereas the 7-hydroxy group with Arg416 and the keto group at the 4-position showed two bonding interactions with Glu258 and Tyr317, respectively (Fig. 6a; Table 2). On the other hand, SR-1 exhibited nine molecular interactions with the certain residues, which include the 3-OH group with Lys236 and one pi–pi interaction with His199, the 5-oxygen group interaction with Ser237, the 11-oxygen atom formed two interactions with Ile175 and Tyr526, the 8-oxygen atom formed interactions with Tyr526 and Arg416, and the 9-oxygen atom displayed two bonded interactions with Tyr317 and Glu258 (Fig. 6b). Furthermore, the physiochemical properties and the toxicity measurements were assessed using Lipinski's rule of five through in silico experiments (Table 3). The bioavailability of the isolates was confirmed as the molecular weight was found to be less than 500, H-bond donors were found to be less than five, the H-bond acceptors were found to be less than ten, and the partition coefficient (cLogP) was predicted to be less than five. These in silico experiments revealed that both the isolates were non-toxic in terms of mutagenic as well tumorigenic properties and beneficial as antiviral agents for treating viral infections.

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4. Conclusions

In the current study, we have successfully isolated and characterized two flavonoids, namely, maackiain (SR-1) and echinoisoflavanone (SR-2), from Sophora interrupta Bedd. root extract and their antiviral properties as well as their molecular mechanisms were evaluated. The results showed that both the isolated compounds have comparatively good in vitro antiviral activities than the standard antiviral drug, Ribavirin. In particular, compound SR-2 was found to be more potent than maackiain and Ribavirin against NDV in vitro. Also, these compounds inhibit viral adsorption, replication, and transcription, thus demonstrating their potential therapeutic effects not only in birds but also against other paramyxoviruses that infect humans and animals. Further efforts for exploring their in vivo antiviral activity and their pharmacokinetics are underway, which will aid in developing them as potential antiviral drugs.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The author WR thanks the Council of Scientific and Industrial Research (CSIR), New Delhi and the authors CB, AR, and PR are grateful to UGC-BSR, New Delhi for financial assistance under CSIR-MRP and UGC-RFSMS (BSR) programs. Furthermore, all the authors are thankful to National Institute of Animal Biotechnology (NIAB), Hyderabad for providing laboratory facilities to carry out the viral studies.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra01820a

This journal is © The Royal Society of Chemistry 2020