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. 2021 Feb 5;11(3):115. doi: 10.1007/s13205-021-02654-6

Phytochemical profiling and cytotoxic evaluation of Premna serratifolia L. against human liver cancer cell line

Chandrashekhar Singh 1, Sumit Kumar Anand 2, Kavindra Nath Tiwari 1,✉,#, Sunil Kumar Mishra 3,✉,#, Poonam Kakkar 2,✉,#
PMCID: PMC7865065  PMID: 33604231

Abstract

Premna serratifolia L. (Lamiaceae) is a medicinal plant, widely distributed in the tropical and subtropical regions and commonly used in traditional medicine. The current study was focused to evaluate the cytotoxic potential of aqueous extract of root of P. serratifolia (AEPS) against human hepatoblastoma cancer cell line (Hep G2).The yield of the dried extract was 5.8% and used for further studies.Cytotoxic potential of AEPS was analyzed by MTT assay, which exhibits IC50 value 1000 µg/mL after 48 h incubation. Hoechst and AO/EtBr staining, ROS measurement, mitochondrial membrane potential, clonogenic and wound healing assays also confirmed the cytotoxic efficacy of AEPS in dose and time-dependent manner. UPLC–Q-TOF–MS/MS analysis of AEPS confirmed the presence of 12polyphenolic compounds, namely 4-hydroxy-3-methoxycinnamic acid, linarin, peonidin-3,5-O-di-beta-glucopyranoside, diosmin, trans-cinnamic acid, daidzein, saponarin, homoorietin, acacetin, sarsasapogenin, phytol and sissotrin. The cytotoxic potential of AEPS might due to presence of biologically active polyphenolic compounds.

Keywords: Premna serratifolia, Human hepatoblastoma cancer cell line, Cytotoxicity, UPLC–Q-TOF–MS/MS

Introduction

Plants have been used to treat various human diseases since ancient times. Most of the drugs used for the treatment of various types of cancer contain several molecules either isolated from plants or synthetic in nature (Courdavault et al. 2020). Plant extracts have been reported earlier showing good efficiency in the management of cancer due to presence of several bioactive compounds (Kumar et al. 2019; Eswaraiah et al. 2020). Several phytoconstituents are helpful in the co treatment and prevention of hepato carcinoma (HC).

Premna serratifolia L. (Lamiaceae) is an important medicinal tree found in various part of the world. In herbal medicines root of the plant was used as a key ingredient. Roots have various medicinal properties and used for the treatment of diabetes, chyluria, inflammation, swelling, headache, bronchitis, liver complication and fever. Pharmacological studies of P. serratifolia confirmed its anti-inflammatory, antimicrobial, hepatoprotective, antioxidant activity and antidiabetic (Singh et al. 2018) activities. Preliminary cytotoxic activity of the P. serratifolia leaf methanol extract have been evaluated against liver (Hep G2), lung (A549) and breast (MCF7) cancer cell lines by MTT assay (Selvam et al. 2012). Alcoholic extract of leaf also exhibited significant in-vitro cytotoxic activity against Ehrlich ascites carcinoma cell lines (Vadivu et al. 2009). On the basis of literature reports phytochemical content in the leaf (Singh et al. 2018), wood (Woo et al. 2019a,b) and root bark (Yadav et al. 2010) of the plant was reported by different workers. UPLC-Q-TOF–MS/MS data analysis revealed the presence of 12polyphenolic compounds in AEPS. Out of 12polyphenolics, 11 first time reported in the root, while phytol was reported earlier by Singh et al. (2011) in the root. Eleven phytochemicals, namely 4-hydroxy-3-methoxycinnamic acid, linarin, peonidin-3,5-O-di-beta-glucopyranoside, diosmin, trans-cinnamic acid, daidzein, saponarin, homoorietin, acacetin, sarsasapogenin and sissotrin were first time reported from aqueous root extract of P. serratifolia.

There is no report of cytotoxicity studies of root extract of P. serratifolia. Therefore, the present report was focused on the evaluation of in vitro cytotoxic activity of aqueous root extract of P. serratifolia against Hep G2 cell using MTT, hoechst and AO/EtBr staining, ROS measurement, mitochondrial membrane potential, clonogenic and wound healing assays. The qualitative analytical method UPLC-Q-TOF–MS/MS was used for tentative identification of bioactive compounds present in the extract.

Materials and methods

Materials

Berberine, 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT), 2′,7′- dichlorodihydrofluoresceindiacetate (DCFH-DA), hoechst 33,258, dihydroethidium (DHE), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine Iodide (JC-1) and fluorescein diacetate (FDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Liver cancer cell line (Hep G2), was procured from American Type Culture Collection (ATCC; Manassas, VA, USA) while, primary hepatocytes (PH) were isolated from rat using collagenase perfusion method (Moldeus 1978) used as a normal cell (control).

Roots of P. serratifolia were obtained from Ayurvedic Garden of Department of Dravyagun, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi. A voucher specimen (BSI/CRC/2016–17) was deposited in the herbarium of Botanical Survey of India (BSI), Allahabad, India after authentication under the accession number 97,879. Fresh roots were washed thoroughly several times under running tap water and further rinsed with distilled water. Roots were chopped into small pieces and 25 g root heated at 60 °C in 100 mL deionized water for 20 min, and after cooling it was filtered through Whatman No.1 filter paper (pore size 25 µm). The yield of aqueous extract of root of P. serratifolia (AEPS) was 5.8%, and it was kept in refrigerator at 4 °C for further studies.

Chemical profiling by UPLC-Q-TOF–MS/MS

Chromatographic analysis of root extract was performed on an UPLC system (Waters, Milford, MA, USA) equipped with a diode array detector, a quaternary solvent delivery system and a column temperature controller. The column temperature was fixed at 25 °C for a BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of water with 0.1% formic acid (HPLC grade, eluent A), 100% acetonitrile (HPLC grade, eluent B) and 100% methanol (HPLC grade, eluent C). Gradient elution of the analyte was carried out using the following program: initial composition of B:C was 90:10% and increased to 80:20% in 2 min, 50:50% for 1–3 min, 30:70% for 3–6 min, then 10:90% for 1 min and finally, decreased quickly to 90:10% for 7–10 min. The flow rate was 0.3 mL/min and the injection volume was 5 µL. Mass spectroscopic analysis of the chemical compounds present in the sample was operated in both positive and negative ion modes equipped with electrospray ionization (ESI) source. The MS parameters were as follows: cone and desolvation gas flow was 52 L/h and 647 l/hr, respectively; source and desolations temperature was 40 °C and 450 °C, respectively. The capillary and cone voltage was set at 2.72 kV and 40 eV, respectively. Mass spectra were recorded across the range of m/z 50–1500. Agilent Mass Hunter Workstation Acquisition Software Version B.05.01 and Qualitative Analysis Software Version B.07.00 were utilized for system control, data acquisition, and data processing. Micro-channel plate (MCP) was functional at 1750 V and Q-TOF mass spectrometer was conducted in MSE mode with low collision energy set at 6 eV in the first function and a collision energy ramp from 20 to 40 eV in the second function. Centroid mode data were collected over the m/z range 100–1000 in both functions, and the scan time was 1 s with an inter scan delay of 0.024 s. The accurate mass and molecular formula denomination were acquired with the Mass Lynx 4.1 software (Waters MS Technologies).

Cytotoxicity studies

MTT assay

Cell cytotoxicity of AEPS treated primary rat hepatocytes as a control and hepatoma cells (Hep G2) was assessed by tetrazolium dye MTT reduction assay as per protocol described by Mosmann (1983). Primary rat hepatocytes are very similar to human liver physiology and it is accepted worldwide as working model to use as a normal cell (control) for toxicity studies (Samodien et al. 2020). In brief, cells were plated in 96-well plate (1 × 104 cells/well) and treated with different concentration (1 × 10–3 − 1 × 103 µg/mL) of AEPS for different time periods (24 h & 48 h) in a dose-dependent manner. After completion of incubation period, media was replenished; MTT (0.5 mg/mL) was added and incubated for 4 h. Thereafter, media was aspirated and 200 μL of DMSO was added to dissolve the formazan crystals and plate was read at 570 nm using a Spectramax PLUS 384 microplate reader (Soft Max Proversion 5.1; Molecular devices, USA). Effect of AEPS on Hep G2 cell viability was calculated and compared to control Hep G2 (assumed to be 100% viable).

Hoechst staining

During apoptosis, chromatic condensation took place and it was studied by Hoechst staining (Zakaria et al. 2009). Approximately, 1 × 105 Hep G2 cells were seeded on cover slips in 6-well plate treated with AEPS (10 and 1000 µg/mL) and incubated for 24 h. After incubation, cells were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde for 30 min at room temperature. Further, cells were washed twice with PBS and then incubated with methanol (chilled, − 20 °C) for 15 min at room temperature followed by 2–3 time washing with PBS. Hoechst 33,258 (10 μg/mL) was added in each well for 10 min and further washed twice with PBS. Finally, cells were examined under fluorescent microscope (Nikon Eclipse 80i, Tokyo, Japan). Brighter fluoresced, condensed and fragmented nuclei are characteristics of cells undergoing apoptosis. Cell numbers and cell nuclei were examined from ten randomly selected areas on each sample at 40 X magnification.

Acridine orange/ ethidium bromide (AO/EtBr) staining

Apoptosis was assessed using AO/EtBr fluorescence double staining assay (Cohen and Scully 1986). Cells approximately 2 × 106 per well in 6-well plate were treated with AEPS (10 and 1000 µg/mL) for 24 h in a dose-dependent manner and one group remains untreated considered control group. After completion of treatment, medium was removed and the cells were washed twice with PBS, followed by acridine orange and ethidium bromide (100 mg/mL AO and 100 mg/mL EtBr in Milli-Q water) double-staining procedure as per protocol. Processed cells were mounted with mounting media containing DAPI and were visualized under EVOS cell imaging system (Life technologies).

Measurement of cellular reactive oxygen species

Intracellular reactive oxygen species (ROS) was measured using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) (Hempel et al. 1999). DCFH-DA is cell-permeable ester; upon hydrolysis by intra cellular esterase it converted into DCFH and retained. DCFH is non fluorescent and further converted into fluorescent DCF after oxidation by ROS and other peroxidase. Estimation of ROS and other peroxidase was done by measuring fluorescence intensity among control and treated cells. Fluorescein diacetate (FDA), a cell-permeant oxidant-insensitive esterase substrate, was included in a parallel experiment that can serve as a viability probe. Cells (1 × 104/well) were seeded in 96 black-well plate and treated with or without AEPS for 24 h and incubated with 10 mM DCFH-DA or 10 mM FDA for 30 min at 37 °C. Fluorescence intensity was detected by spectrofluorometer (Varioskan Flash, Thermo) at excitation and emission wavelengths of 485 nm and 530 nm, respectively. Superoxide generation in treated cells was assessed by dihydroethidium (DHE). Cells were seeded with cover slips in a 6-well plate (1 × 10 cells per well). After the treatment, DHE is added to control and treated cells at 5 mg/ml, incubated up to 30 min. After incubation, cells were washed thrice with PBS and mounted with mounting media containing DAPI. Microscopic analysis was performed to assess superoxide generation.

Determination of mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨm) was assessed using JC-1(5, 5′, 6, 6′ tetrachloro-1, 1′, 3, 3′-tetraethyl-benzimidazolylcarbocyanine iodide), a lipophilic cationic dye, which enters selectively in mitochondria. In polarized mitochondria, JC-1 forms aggregates inside mitochondria and emits red fluorescence. In polarized state of mitochondria it remains in monomeric forms and emits green fluorescence. Briefly, monolayer cultures (1 × 104 cells) were grown into a 96-well plate and treated with vehicle and AEPS for 24 h followed by incubation with 5 µM JC-1 for 10 min in dark. Fluorescence intensity was monitored on a spectrofluorometer (Varioskan Flash, Thermo) at excitation and emission wavelengths of 520 nm and 596 nm, respectively (Koppikar et al. 2010).

Clonogenic assay

As cancer cells have the property of unregulated growth, cell proliferation and no contact inhibition, they divide rapidly and form colonies of cancer cells. To examine the effect of AEPS on colony formation, clonogenic assay was performed. Hep G2 cells were incubated overnight in 24-well plate with 1 × 103 cells per well. Further it was treated with AEPS (10 and 1000 µg/mL) for 24 h. After the completion of treatment, media was replaced with fresh media and cells were allowed to grow in Dulbecco's Modified Eagle's Medium (DMEM) for 1 week to divide rapidly and form colonies of the cells. Culture media was changed at regular interval of 2 days. When cells get confluence between 70 and 80%, culture media was removed and washed twice with PBS. Fixing solution (Acetic acid: methanol, 7:1) was used for 5 min at room temperature for fixation of groups of colonies. Further cells were stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. The colony counting was done; sufficiently large colonies were observed in both treated and control cells (Franken et al. 2006).

Wound healing assay

Wound healing assay was used to monitor the migration and growth of cells towards wound. In present communication, wound healing assay was performed to assess the effect of AEPS on migration potential of cancer cells (Rodriguez et al. 2005). Hep G2 cells were maintained as monolayer and grown in a humidified 5% CO2 environment at 37 °C. Cells were seeded in a 6-well plate and allowed to grow until reaching 90% confluency per well. When cell monolayer reached their confluency mark, scratch was made with a 1000-µL pipette. Further, scratched wells were treated with AEPS (1000 µg/mL) for 24 h. After completion of treatment, width of the scratch was measured under phase contrast microscope (10 X) at 0 h, 12 h and 24 h. Migration distance or wound healing distance was measured using Image J software and plotted with their normalization with control.

Statistical analysis

The results were expressed as Mean ± SD and analyzed by one-way analysis of variance at P < 0.05; P < 0.01; P < 0.001 was considered a significant difference.

Results and discussion

Identification of major compounds present in AEPS using UPLC-Q-TOF–MS/MS

The UPLC-Q-TOF–MS/MS total ion chromatogram (TIC) of AEPS in both positive and negative ion mode was shown in Fig. 1a and b respectively. AEPS was profiled for the presence of bioactive compounds by their mass through UPLC–Q-TOF–MS/MS. These compounds were tentatively identified based on their retention time, ESI [M-H−], MS/MS and m/z base ions. The compounds were tentatively identified by using the combination of MS and MS/MS data, information available in the literature and established database (http://spectra.psc.riken.jp/). The compound was considered as its derivatives if the product ion spectrum did not hit any of the product ion m/z in the search but hit only the first and one or more of the product ion m/z in the spectrum. Data revealed that AEPS contained 12polyphenolic compounds. Among these polyphenolics one belongs to phenolic acid (4-hydroxy-3-methoxycinnamic acid), five flavones (linarin, diosmin, saponarin, homoorietin,acacetin), one anthocyanin (peonidin-3,5-O-di-beta-glucopyranoside), two tepenoids (sarsasapogenin, phytol), two isoflavones (daidzein, sissotrin) and one phenyl propanoid (trans-cinnamic acid) categories as detailed given in the Table 1. On the basis of previous studies these pure compounds were isolated from different plants, showed different pharmacological activities.

Fig. 1.

Fig. 1

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Total ion chromatogram of aqueous root extract of P. serratifolia (a) in + ve ion mode showing peaks of identified compounds (1) 4-hydroxy-3-methoxycinnamic acid, (2) linarin, (3) peonidin-3, 5-O-di-beta-glucopyranoside, (4) diosmin and (5) trans-cinnamic acid (b) in –ve ion mode showing peaks of identified compounds (1) daidzein, (2) saponarin, (3) homoorietin (4) acacetin, (5) sarsasapogenin, (6) phytol and (7) sissotrin

Table 1.

Major compounds identified in aqueous root extract of P. serratifolia by UPLC/MS–MS

graphic file with name 13205_2021_2654_Tab1_HTML.jpg

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Cytotoxicity assessment

MTT assay is widely used for cytotoxicity assessment. MTT tetrazolium is a dye which depends upon the metabolic activity of the cell. When dye enteres inside the living cells, mitochondria converted into purple farmazon crystals. These crystals are soluble in DMSO and leads to a purple color development. Increase in the absorbance of color indicates about metabolic activity of the cells. Dose response curve for cytotoxicity of AEPS towards primary rat hepatocyte cells (control) were measured using MTT assay (Fig. 2). Cells treated with AEPS in a dose-dependent manner for 48 h. Results suggest the non-cytotoxic nature of AEPS for control cells, which is safe for use up to the dose level of 1000 µg/mL. Hep G2 cells treated with AEPS in a dose-dependent manner for 24 and 48 h. Result revealed that HepG2 cells treated with 1000 µg/mL AEPS caused 54% and 58% cell death after 24 h and 48 h, respectively (Fig. 3a). Treatment induced cell death suggests about cytotoxic efficiency of AEPS. Treatment of Hep G2 cells with AEPS showed significant morphological changes as compared to untreated cells. It caused membrane disruption and shrinkage as observed under bright field microscopy (Fig. 3b). In the present study, AEPS cytotoxicity to Hep G2 cells was observed that is dose and time-dependent manner.

Fig. 2.

Fig. 2

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Dose-response curve for cytotoxicity of AEPS towards primary rat hepatocyte cell was measured by using MTT assay. Cells treated with AEPS in a dose-dependent manner for 48 h.Data are presented as mean ± SD. *P < 0.05 significantly different from the control

Fig. 3.

Fig. 3

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Dose–response curve for cytotoxicity of AEPS towards Hep G2 cell was measured by using MTT assay. Cells treated with AEPS in a dose-dependent manner for 24 h and 48 h (a), Effect of AEPS treatment on cellular morphological changes in Hep G2 cell at different concentration (b). Data are presented as mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 significantly different from the control

Evaluation of oxidative stress

Reactive oxygen species (ROS) generation in pathogenesis is common feature. Different types of free radicals were produced under stress, it caused breakdown of cellular biomolecules like protein and nucleic acids. ROS generation inside cells was assessed by using fluorescent DCFDA and DHE dyes. DHE used for the measurement of total ROS production, while with DCFDA we can get idea about superoxide generation inside cells. There was a significant increase in DHE fluorescence in microscopic analysis in AEPS treated cells (Fig. 4a). DCFDA was done fluorimetrically; increased fluorescence confirmed ROS production in Hep G2 cells (Fig. 4b). AEPS treated cells showed significant increase of fluorescence in DHE and DCFH-DA in comparison to vehicle-treated Hep G2 cells in a dose-dependent manner. These observations showed that AEPS at 1000 µg/mL was good enough to induce ROS generation and apoptotic cell death.

Fig. 4.

Fig. 4

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Intracellular ROS observation by Fluorescent Microscopy. Hep G2 cells stained with nuclear dye (Hoechst 33,258), DHE and the overlapped images after 24 h of AEPS treatment (a). (The representative image of DHE is red, the nuclear dye is blue), fold change in DCFH-DA (10 mM) after 24 h of AEPS treatment (b). Data are presented as mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 significantly different from the control

Mitochondrial membrane potential (MMP) assessment

Mitochondria were affected by the reactive oxygen species generated inside the cells. Mitochondrial membrane potential was assessed by FACS analysis by using MMP specific JC-1 dye. In the absence of the ROS inside healthy cells and mitochondria, JC-1 generally remains in aggregated form in mitochondria. Under stressed conditions, mitochondrial membrane potential affected which caused its depolarization. In this situation, the monomeric form of JC-1 comes out from mitochondria into cytosol and fluoresce red to green. Increase in the fluorescence from red to green indicates mitochondrial depolarization. We observed a significant increase in fluorescence intensity in AEPI treated cells which are many fold higher than control cells (Fig. 5). Results revealed that AEPS significantly affects mitochondrial membrane potential of cells and caused depolarization of mitochondria more effectively than control Hep G2 cells.

Fig. 5.

Fig. 5

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Measurement of increase in fluorescence intensity of AEPS (0.001–1000 µg/mL) treated Hep G2 cells by FACS analysis using Mitochondrial membrane potential specific JC-1 dye. Data are presented as mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 significantly different from the control

Clonogenic assay

During clonogenic assay we observed a significant decrease in number of colonies’ formation in AEPS treated Hep G2 cells, than control treated cells. Hep G2 cells without AEPS treated showed good colonies formation (Fig. 6). Result suggests that AEPS was effective for inhibition of cancer cells division and colonization.

Fig. 6.

Fig. 6

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Assessment of colony formation potential of Hep G2 cells after treatment with AEPS (10 and 1000 µg/mL) for 24 h

Wound healing assay

Cancer cells have the properties of unregulated cell division and they can fill empty space in a very short time. They divide rapidly to fill the scratch and heal the wound (Fig. 7a). We observed a significant decrease in wound healing in AEPS (1000 µg/mL) treated Hep G2 cells in comparison to control (Fig. 7b). It is also cleared that AEPS effectively inhibits the wound healing by reducing the migration potential of cancer cells.

Fig. 7.

Fig. 7

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Wound healing potential of Hep G2 cells as observed under phase contrast microscope after treatment of AEPS (1000 µg/mL) for 24 h (a) and fold change in wound healing potential of AEPS at 0, 12 and 24 h (b). Data are presented as mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 significantly different from the control

Apoptosis induction

Anticancer drugs target the apoptotic pathway which leads to cell death. In this study, we observed mitochondrial dysfunction and significant decrease in mitochondrial membrane potential (MMP) which indicates involvement of intrinsic pathway of apoptotic cell death. There was a significant increase in Hoechst fluorescence in AEPS (10 and 1000 µg/mL) treated HepG2 cells in comparison to control (Fig. 8a). It showed that AEPS has the potential to induce apoptotic cell death in treated Hep G2 cells. We have confirmed apoptosis inducing ability of AEPS by AO/EtBr dual staining. In this staining method we used two fluorescent dyes named acridine orange and ethidium bromide, which stain the nucleus of the cells. We observed significant changes in the cell morphology after AO/EtBr staining in AEPS (1000 µg/mL) treated and untreated (control) Hep G2 cells. Image(Fig. 8b) showed that untreated Hep G2 cell (control) nuclei were uniformly green and orange-stained apoptotic bodies’ formation was not found there, while in AEPS treated cells’ nuclear condensation and orange-stained apoptotic bodies were significantly higher than control Hep G2 cells. We observed significant changes in nuclear condensation in AEPS treated Hep G2 cells (Fig. 8b) in comparison to untreated Hep G2 cells with healthy nuclei. All these observation confirmed that AEPS have the potential of inducing apoptotic cell death which is mitochondria mediated and is more effective in inducing apoptotic cell death in cancer Hep G2 cells than control.

Fig. 8.

Fig. 8

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Fluorescent microscope analysis of apoptosis-inducing potential of AEPS in Hep G2 (10 and 1000 µg/mL) cells after Hoechst and AO/EtBr staining. Analysis of nuclear condensation and morphology of hoechst stained Hep G2 cell treated with AEPS (a) and apoptosis of Hep G2 cell after AO/EtBr staining (b)

Conclusion

Aqueous root extract of P. serratifolia demonstrated significant cytotoxicity activity against human hepatoblastoma cell line, while it is non-cytotoxic to primary rat hepatocytes. UPLC-Q-TOF–MS/MS analysis tentatively identified 12 major polyphenolic compounds present in AEPS. The cytotoxic potential of AEPS may be due to presence of these polyphenolic compounds. It suggests that this extract may be a good source for isolation of cytotoxic compounds. Further study is needed to isolate, characterize and quantify these individual compounds. It will be useful to study the exact mechanism of action of these compounds.

Acknowledgements

Author CS extends their sincere appreciation to the University Grant Commission (UGC), New Delhi, India for financial support in form of Ph.D. Fellowship.

Author contributions

CS: Methodology. SKA: Formal analysis. KNT: Methodology. SKM: Formal analysis. PK: Formal analysis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Ethical clearance was granted by the “Ethical Committee of the Institute of Science, Banaras Hindu University, Varanasi, India (IAEC) (Approval No: F. Sc./88/IAEC/2016–17).

Footnotes

Kavindra Nath Tiwari, Sunil Kumar Mishra and Poonam Kakkar contributed equally.

Contributor Information

Kavindra Nath Tiwari, Email: kntiwaribhu@gmail.com.

Sunil Kumar Mishra, Email: skmishra.phe@iitbhu.ac.in.

Poonam Kakkar, Email: pkakkar@iitr.res.in.

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