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IJCRR - Vol 13 Issue 07, April, 2021

Pages: 209-215

Date of Publication: 12-Apr-2021


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The Anticancer Activity of Artemisia Judaica Crude Extract in Human Hepatocellular Carcinoma HepG2 Cells by Induction of Apoptosis and Cell Cycle Arrest

Author: Neima K. Al-Senosy, Naglaa M. Ebeed, Lamiaa M. Salem, Shenouda M. Girgis, Ekram S. Ahmad

Category: Healthcare

Abstract:Introduction: Cancer is one of the major causes of death around the world. Medicinal plants have been investigated across the world to exploit their potential anticancer activity on a large scale of applications. Objective: The present study is focused on the anticancer activity of the methanolic extract of the medicinal plant, Artemisia Judaica, on Human hepatocellular carcinoma (HepG2) and normal liver (THLE2) cell lines, as well as the mechanisms involved were also investigated. Methods: The collected aerial parts of the plant were extracted by maceration with methanol and the crude extract was collected and stored until use. Cytotoxicity and cell proliferation, cell cycle analysis and the expression level of apoptosis-related genes such as p21, CycB1, CDK1, p53, Bcl-2 and Bax in HepG2 cell lines were estimated using the MTT colourimetric, flow cytometry and quantitative real-time PCR (qRT-PCR) assays, respectively. Results: The results showed that A. Judaica extract (IC50 = 33.76\?g/ml) displayed strong cytotoxicity and antiproliferative effect in HepG2 cancer cells. On the other hand, this extract exhibited no cytotoxic activity on the liver normal cell line (THLE2). Flow cytometric analysis of propidium iodide staining revealed that the treatment of HepG2 cells with A. Judaica led to an increase in G2/M phase cell cycle arrest. The qRT-PCR assay revealed that both cyclin B1 (cycB1) and cyclin-dependent kinase (CDK1) genes, as well Bcl-2 showed down-regulation expression levels in HepG2 treated with A. Judaica compared to the untreated cell line. Furthermore, the apoptotic mechanism activated by the plant extract resulted in up-regulation of p53, P21and Bax at mRNA level on HepG2 cell line. Conclusion: These results suggest that A. Judaica could be a promising candidate species as a natural source of anticancer molecules.

Keywords: Artemisia Judaica, Antiproliferation, Apoptosis, Anticancer, HepG2 cell line

Full Text:

INTRODUCTION

Cancer is one of the major causes of death worldwide.  It is characterized by genetic alterations of normal cells which become malignant cells. Those are characterized by uncontrolled cell growth, immortality, invasiveness, and the ability to form distant metastasis. It was found that natural products may interfere with the carcinogenesis process by altering the behaviour of tumour cell and targeting cancer cells signalling pathways.1

        Among different cancer types, liver cancer is the 4th most common cause of death from cancer worldwide, the incidence of human hepatocellular carcinoma (HCC) is increasing particularly in males in some countries. Hepatitis B and C viruses (HBV and HCV) and dietary aflatoxin intake remain the major causative factors of HCC, where different modes of cancer therapy for HCC have been tried such as surgery, chemotherapy, radiotherapy, as well, tremendous works have been done at the molecular level.2

The use of natural products and supplements of medicinal plants has very increased over the past three decades with more than 80% of people worldwide depend on them for some part of primary healthcare.3 The diet enriched with naturally occurring substances significantly reduces the risk for some cancers. Consequently, many drugs used for the treatment of cancer have been discovered from medicinal plants.4

Biologically active components of the medicinal plants target tumour cells by different mechanisms, resulting in angiogenesis, inhibition of carcinogenesis, cell cycle arrest, oxidative stress, autophagy or differentiation and apoptosis.5-9           Artemisia was found to possess sesquiterpene lactones and other derived phytochemicals as active components. Sesquiterpene lactones were used for their therapeutic and other properties.10 Recently, monoterpenes, sesquiterpenes, sesquiterpene lactones, flavonoids, coumarins, sterols, polyacetylenes have been isolated from Artemisia species.11 Some Artemisia species have shown that it possesses medicinal properties such as anti-bacterial and anti-cancer effects.12 Few species of Artemisia such as Artemisia Judaica, showed promising anticancer activities against the proliferation of cancer cell lines.13 The main effect of its essential oils is attributed to the main constitutes, the thujone, in this species,14  enriched with fraction has potential anticancer activities.15

A. Judaica (Arabic name, Shih Balady) is a perennial fragment shrub that is abundant in North Africa and Middle Eastern countries.16 As well in Saudi Arabia, Yemen and Egypt.17-19 This plant grows abundantly in different parts of the Arabian peninsula such as Saudi Arabia and Yemen,17,18 and has been used in traditional Egyptian medicine for the treatment of gastrointestinal disorders.19 Besides, the Artemisia species have been used in Iranian traditional medicine as an anti-infectious, anti-bacterial, gastric tonic, digestive and stomachic.20 Isolated compounds from A. Judaica have exhibited antiviral, antibacterial, antifungal, and cytoprotective effects,21-23 and for the treatment of hepatitis, cancer and menstrual-related disorders.24 The composition of A. Judaica includes artemisinic acid, sesquiterpene lactones, methyl wormwood, artemisinic alcohol, eucalyptol, Artemisia ketone, camphor, caryophyllene, piperitone and essential oil.25-26 Besides, diverse chemical components, such as flavonoids, coumarins, sterols, polyacetylenes, monoterpenes, polyphenols, sesquiterpenes and sesquiterpenes lactones, have been found in plants from the Artemisia genus.22,24

           Sesquiterpene lactone is also a potent apoptotic inducer in cancer cells via multiple pathways. It is readily depleted intracellular glutathione (GSH), disrupts cellular redox balance, depletes intracellular thiols,27 triggers an intracellular reactive oxygen species (ROS),28 decreases anti-apoptotic Bcl-2 protein expression and induce apoptosis.29 Studies of the molecular mechanisms have shown that caspase and p53-independent activation, downregulation of Bcl-2, generation of ROS, reduction of mitochondrial membrane potential, increased amount of Bax protein, arrest of the cell cycle at G2/M-phase, suppression of the Notch 1, and inhibition of NF-κB are important mechanisms for the cytotoxic effects of Artemisia.30 As well, flow cytometric analysis for cell cycle revealed increment of G2/M phase cell cycle arrest after treatment of HepG2 cells with A. herba alba extract. The apoptotic mechanism was also activated by the crude extract of Artemisia included up-regulation of p53 and Bax and downregulation of Bcl-2 expression levels with no cytotoxic effect for normal cell lines exposed to the plant extract.31

Besides, Artemisia extract increased the number of cells in the G2/M phases, followed by caspase 3 upregulation, and apoptosis. Further, it is inhibited cancer cell proliferation and induced apoptosis.32 Therefore; the present study is designed to evaluate the anticancer activity of crude extract of A. Judaica, on human hepatocellular carcinoma (HepG2) cell line. The possible underlying antiproliferation mechanisms were investigated, by studying the effect of A. Judaica on cytotoxicity, cell cycle arrest and apoptosis-related genes in the human hepatocellular carcinoma (HepG2) cell line.

MATERIALS AND METHODS

Plant collection and extract preparation

The aerial parts (leaves and stems) of A. Judaica were collected during the summer season (July 2020) from Wadi Gharandal, South Sina, Egypt. Taxonomical identification was confirmed by a botanist and voucher specimen, were deposited in the Herbarium of the National Research Center, Dokki, Cairo, Egypt. Herbs were washed and shade dried for a week and was milled to fine powder, then it was extracted with methanol at a ratio of 20 g dry powder in 200 ml of ethanol for 48 h using the maceration method. The liquid extract was filtered and concentrated under vacuum by soxhlet extraction, then stored in the dark at 4°C until use.                           

Assessment of cytotoxic and anti-proliferous activities of A. Judaica

Cell proliferation and viability of the cells were estimated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colourimetric assay. Human hepatocellular carcinoma (HepG2) and normal liver (THLE2) cell lines in exponential growth phase were seeded at a density of 1 x 104 cells per well (100 μl/well) onto a 96-well plate (Falcon, Franklin Likes, NJ, USA) in DMEM medium (GIBCO, Grand Island, New York, USA; Cat.no.A1049101). The cell density was adjusted by the trypan blue exclusion method. The whole compounds were cultured in different concentrations (ranged from 0 to 200 µg/mL) for 24 hours at 37 °C in a 5% CO2 with a 95% humidity incubator. Besides, different concentrations of cisplatin as a reference chemotherapeutic drug were added and the microplates were incubated for a further 48 hour in DMEM medium (200 µL). The medium was washed gently twice with ice-cold PBS and a volume of 200 μL MTT  [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a yellow tetrazole, (Molecular probes, Eugene, Oregon, USA; Cat.no.V-13154)] was added to each well. The microplate was incubated at 37 °C for another 4 hours in a CO2 incubator. About 180 μL medium/MTT was removed and 100 μL of acidified isopropanol were added per well to solubilize the formazan produced. Finally, the microplate was incubated with shaking for 15 minutes. The absorbance of each well was measured at 630 nm using a microplate reader (ELX800,  Biokit, Spain). Assays were performed in triplicate on three independent experiments. Sigmoidal and dose-dependent curves were constructed to plot the results of the experiment. The concentration of the compounds inhibiting cancer cell growth by 50% of the control level (IC50) was calculated using this sigmoidal curve.

Cell cycle analysis by flow cytometry

To evaluate the effect of A. Judaica crude extract on the cancerous cell division of HepG2, the cell cycle analysis was carried out using the protocol of Applied Bio-system, USA. Cells were digested with warm Trypsin-EDTA + warm PBS-EDTA (0.25%) (500 µl + 500 µl) with incubation for 10 minutes at 37°C. The mixture was centrifuged at 450 rpm for 5 min, and then the supernatant was carefully removed. The mixture was washed twice in warm PBS and the cell pellet was re-suspended in 500 µl warm PBS, centrifuged and the supernatant was removed. A volume of 150 µl PBS + 350 µl ice-cold 70% ethanol was added and incubated at 4°C for 1 hour to fix the cells. To remove ethanol, the mixture was centrifuged at 350 rpm for 10 minutes and then the supernatant was carefully removed. The mixture was washed twice in warm PBS and the cells were re-suspended in 500 µl warm PBS, centrifuged and the supernatant was removed. The cells were re-suspended in 100 µl PBS and stored at 4° for up to 4 days. In the darkness, the cells were stained with 100 µl of propidium iodide (PI) solution + 50 µl RNase A solution (100 µg/ml) and incubated in darkness for 30-60 min. The stained cells were read in Attune flow cytometer (Applied Bio-system, USA).

Determination of the expression levels of apoptosis-regulatory genes by quantitative real-time PCR

RNA extraction was carried out utilizing an RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) for HepG2 cell lines, or treated with IC50 of A. Judaica extract for 24 h. The procedure was performed according to the manufacturer’s instructions. The cDNA was synthesized from purified RNA with an RT2 First Strand Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s guidelines as the template for RT-qPCR. Corbett Rotor-Gene 6000 (Qiagen, Inc., Valencia, CA, USA) was used to perform quantitative real-time reverse transcriptase PCR (qRT-PCR). A final volume of 25 µl pre-mix was prepared to contain 12.5 µl of RT2 SYBR R Green ROXTM FAST master mix (Qiagen, Inc., Valencia, CA, USA), 1 µl of primers (RT2 qPCR Primer Assays, Qiagen, Inc., Valencia, CA, USA), 1 µl of cDNA, and 10.5 µl RNase-free water to make the final volume. The following primer pairs for target genes and the housekeeping β-actin gene was chosen from the Primer Bank website.

The default PCR conditions were as follows: the PCR plate was run at 95?C for 10 min to activate the enzyme, 40 cycles of 15 s at 95?C (denaturation) followed by 30 s at 60?C (annealing and synthesis). Finally, the dissociation curve was constructed immediately after the PCR run to check and verify results. The housekeeping gene β-actin was used as a reference to calculate fold change in target gene expression.

Statistical Analysis

Data were expressed as means ± standard error (SEM). One way ANOVA using SPSS 18.0 software was used to detect the significant difference. Values were considered statistically significant when P≤0.05. Comparison of means was carried out with Tukey’s HSD test.

RESULTS

Cytotoxic activity of A. Judaica crude extract using MTT assay

 The cytotoxic activity of A. Judaica crude extract was screened on human hepatocellular carcinoma cell line (HepG2) by MTT assay (Fig. 1). The results revealed that A. Judaica extract (IC50 = 33.76μg/ml), inhibited the proliferation and exhibited increasing in cytotoxic activity to HepG2 cell line in a dose-dependent manner, where, the percentage of cell viability declined up to 20% by A. Judaica dose increment, while the crude extract of A. Judaica didn’t induce growth inhibitory effect on normal liver cell line (THLE2) (IC50 = 1041μg/ml), where, a very low cytotoxic activity was noticed by A. Judaica against normal liver cell line.

Effect of A. Judaica crude extract on cell cycle arrest using flow cytometry

The DNA content in A.judaica treated HepG2 cells was assessed using the cell cycle phase distribution analysis. The cell cycle arrest was analyzed using flow cytometry (Fig. 2). According to the inhibition rate of liver cancer (HepG2) cell viability, the results revealed that the anti-proliferative effect of A. Judaica was associated with cell cycle arrest using flow cytometry-based cell cycle distribution (Table 1). Compared to the control group, the treatment with A.judaica crude extract at 33.76 μg/ml affected the cell cycle distribution on HepG2 cells, where, the G0/G1 phase showed decreased from 60% to 29% of cell cycle arrest, while the S-phase percentage increased slightly from 19% to 22% in the HepG2 and HepG2 treated with A.judaica crude extract, respectively. Interestingly, the percentage of HepG2 cells at the G2/M phase was increased (49%) compared to (21%) for the untreated cell line. These results suggested that A. Judaica inhibited the cellular proliferation of HepG2 cells via G2/M phase arrest of the cell cycle.

The expression level of apoptosis-related genes modulated by A. Judaica

 To evaluate the molecular mechanism of A. Judaica-induced apoptosis in HepG2 cells, the expression level of apoptosis-related genes such as p21, cyclin B1 (CycB1), cyclin-dependent kinase (CDK1), p53, Bcl-2 and Bax in HepG2 cells (Fig. 3) was estimated by quantitative real-time PCR (qRT-PCR). Results revealed that compared to the untreated HepG2 group, the expression level of p21, p53 and Bax was increased (upregulated) in A.judaica treated HepG2 cell line, whereas, the expression level of Bcl-2, CycB1 and CDK1genes was down-regulated (decreased) in HepG2 treated with A. Judaica compared to the untreated HepG2 cell line.

DISCUSSION

Medicinal herbs have shown their pharmacological activities as a new drug reaching the market.33,34 Different forms of cancer are constantly gaining resistance to current drugs, creating a need for the discovery of new drugs. A. Judaica extract showed remarkable cytotoxic activity against the tested liver cancer cell lines in a dose-dependent manner. This could be due to the presence of artemisinin and other sesquiterpene lactones, which have been found in A. Judaica.35,36 Artemisinin which is a sesquiterpene lactone demonstrated antitumor agent and anticancer activity when tested in vitro and in vivo.37,38

These results agree with previous studies reporting biologically active compounds from the medicinal plant A. Judaica possess sesquiterpene lactones, essential oils and other derived phytochemicals as active constituents which have shown medicinal properties such as anticancer activities (cytotoxicity and antiproliferation) for cancer cell lines, with no cytotoxic effect for normal cell lines exposed to the plant extract.31

The cell cycle arrest was analyzed using flow cytometry-based cell cycle distribution. The results indicated that A. Judaica extract inhibited the liver cancer (HepG2) cell cycle through triggering G2/M phase arrest involved transcriptional suppression, where the percentage of HepG2 cells at the G2/M phase was increased and reached about 50 % (49% ± 2.08) compared to 21% for the untreated cell line. That coincided with Lang et al.,32 who found that Artemisia extracts induced accumulation of multinucleated cancer cells within 24 h of treatment, increased the number of cells in the S and G2/M phases of the cell cycle, followed by loss of mitochondrial membrane potential, caspase 3 activation, and formation of an apoptotic cell population. These data indicated that A. Judaica exhibited an antiproliferative effect by a cell cycle blocking at the G2/M phase and apoptosis mediated cytotoxicity in carcinoma cells. However, Honda et al.,39 found that equipotent concentrations of A.judiaca extract highly arrested the cell cycle in the G0/G1 phase of cancer cell lines.

The current study inspected the ability of A.judiaca crude extracts to induce apoptosis where it is known to be the most promising pathway for a cancer therapy strategy. Consequently, results showed that the extract had the highest cytotoxic activity, have ingredients or molecules involved in the activation mechanisms of one or more antiproliferative pathways. A. Judaica medicinal plant induces apoptotic process which is modulated by different tumour suppressor genes including p53. The P53 induces apoptotic cell death by direct or indirect change expression of the Bcl-2 family of proteins, Bcl-2 and Bax.40 The Bcl-2 gene is an anti-apoptotic gene that suppresses initiation steps of apoptosis via inhibition of the pro-apoptotic proteins.41 P53 may modulate susceptibility of cells to apoptosis by downregulation of BCL-2 and causing up-regulation of BAX 27 and that coincides with our findings.

The cell cycle is mediated by the activation of a highly conserved protein kinase family, the cyclin-dependent kinases (CDKs).42 Cyclins can activate CDKs by forming complexes with Cdks, and these cyclin/CDK complexes are cell cycle regulators. Among them, the cyclin B1/CDK1 complex, in which B-type cyclins associate with CDK1, is considered as one of the primary regulators of transition from the G2 to M phase. This complex was originally discovered and defined as the maturation-promoting factor or M phase-promoting factor (MPF).43 In our study, the down-regulation of CycB1, Bcl-2 and CDK1 gene expression as responsible genes for Cyclins and cyclin-dependent kinase (the cell cycle regulators cyclin/CDK complexes) in HepG2 treated with A. Judaica compared to the untreated HepG2 cell line at G2/M phases of the cell cycle, confirm these findings.

Sesquiterpene  lactone as active constituents of Artemisia was found to be a potent apoptotic inducer in cancer cells via multiple pathways especially in depletes intracellular GSH, disrupts cellular redox balance, depletes intracellular thiols, 27 triggers an intracellular reactive oxygen species that lead to mitochondrial dysfunction: loss of mitochondrial membrane potential, the onset of mitochondrial membrane transition, and release of mitochondrial pro-apoptotic proteins,28 decreases anti-apoptotic Bcl-2 protein expression and induce apoptosis.29

CONCLUSION

Based on the findings of the study, the crude extract of A. Judaica was found to had anticancer properties specific for human tumour cells, without any toxicity on normal cells. These results indicated that A. Judaica has an antiproliferative effect on HepG2 cell lines.  Moreover, A. Judaica crude extract evaluated to identify the mechanisms behind the toxicity such as cell cycle blocking at the G2/M phase using flow cytometry. The real time-PCR to measure the mRNA levels of cyclin B1 and cyclin-dependent kinase (CDK1) genes (cell cycle regulator genes) and Bcl2, where those genes showed down-regulation expression levels in HepG2 treated with A. Judaica compared to the untreated cell line. Gene expression of ribonucleic acid (RNA) study confirms A. Judaica extract to induction of apoptosis via activation of p53, p21 and Bax and inactivated of Bcl2. Taken together, these data suggest that A. Judaica could be a promising candidate species as a source of anticancer molecules. Finally, natural products such as A.judaica are attractive sources for the development of new medicinal and therapeutic agents. Those with anticancer potential and apoptosis may be more selective and have weaker adverse effects on normal cells compared to chemotherapy used for cancer treatment. So, these research trends indicate that natural products will be among the most important sources of new drugs for cancer prevention and treatment in human being.

Conflicts of Interest: The authors declare that they have no conflicts of interest related to this study. This work was financially supported by us.

Acknowledgements: Authors acknowledge the immense help received from the scholars whose articles are cited and included in references to this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

Source of funding: We expend from our account.

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Best Article Award

A Study by Humaira Tahir et al. entitled "Comparison of First Analgesic Demand after Major Surgeries of Obstetrics and Gynecology between Pre-Emptive Versus Intra-Operative Groups by Using Intravenous Paracetamol: A Cross-Sectional Study" is awarded Best Article for Vol 14 issue 14
A Study by Monica K. entitled "Risk Predictors for Lymphoma Development in Sjogren Syndrome - A Systematic Review" is awarded Best Article for Vol 14 issue 13
A Study by Mokhtar M Sh et al. entitled "Prevalence of Hospital Mortality of Critically Ill Elderly Patients" is awarded Best Article for Vol 14 issue 12
A Study by Vidya S. Bhat et al. entitled "Effect of an Indigenous Cleanser on the Microbial Biofilm on Acrylic Denture Base - A Pilot Study" is awarded Best Article for Vol 14 issue 11
A Study by Pandya S. et al. entitled "Acute and 28-Day Repeated Dose Subacute Toxicological Evaluation of Coroprotect Tablet in Rodents" is awarded Best Article for Vol 14 issue 10
A Study by Muhammad Zaki et al. entitled "Effect of Hemoglobin Level on the Severity of Acute Bronchiolitis in Children: A Case-Control Study" is awarded Best Article for Vol 14 issue 09
A Study by Vinita S & Ayushi S entitled "Role of Colour Doppler and Transvaginal Sonography for diagnosis of endometrial pathology in women presenting with Abnormal Uterine Bleeding" is awarded Best Article for Vol 14 issue 08
A Study by Prabhu A et al. entitled "Awareness of Common Eye Conditions among the ASHA (Accredited Social Health Activist) Workers in the Rural Communities of Udupi District- A Pilot Study" is awarded Best Article for Vol 14 issue 07
A Study by Divya MP et al. entitled "Non-Echoplanar Diffusion-Weighted Imaging and 3D Fiesta Magnetic Resonance Imaging Sequences with High Resolution Computed Tomography Temporal Bone in Assessment and Predicting the Outcome of Chronic Suppurative Otitis Media with Cholesteatoma" is awarded Best Article for Vol 14 issue 06
A Study by Zahoor Illahi Soomro et al. entitled "Functional Outcomes of Fracture Distal Radius after Fixation with Two Different Plates: A Retrospective Comparative Study" is awarded Best Article for Vol 14 issue 05
A Study by Ajai KG & Athira KN entitled "Patients’ Gratification Towards Service Delivery Among Government Hospitals with Particular Orientation Towards Primary Health Centres" is awarded Best Article for Vol 14 issue 04
A Study by Mbungu Mulaila AP et al. entitled "Ovarian Pregnancy in Kindu City, D.R. Congo - A Case Report" is awarded Best Article for Vol 14 issue 03
A Study by Maryam MJ et al. entitled "Evaluation Serum Chemerin and Visfatin Levels with Rheumatoid Arthritis: Possible Diagnostic Biomarkers" is awarded Best Article for Vol 14 issue 02
A Study by Shanthan KR et al. entitled "Comparison of Ultrasound Guided Versus Nerve Stimulator Guided Technique of Supraclavicular Brachial Plexus Block in Patients Undergoing Upper Limb Surgeries" is awarded Best Article for Vol 14 issue 01
A Study by Amol Sanap et al. entitled "The Outcome of Coxofemoral Bypass Using Cemented Bipolar Hemiarthroplasty in the Treatment of Unstable Intertrochanteric Fracture of Femur in a Rural Setup" is awarded Best Article Award of Vol 13 issue 24
A Study by Manoj KP et al. entitled "A Randomized Comparative Clinical Trial to Know the Efficacy of Ultrasound-Guided Transversus Abdominis Plane Block Against Multimodal Analgesia for Postoperative Analgesia Following Caesarean Section" is awarded Best Article Award of Vol 13 issue 23
A Study by Karimova II et al. entitled "Changes in the Activity of Intestinal Carbohydrases in Alloxan-Induced Diabetic Rats and Their Correction with Prenalon" is awarded Best Article of Vol 13 issue 22
A Study by Ashish B Roge et al. entitled "Development, Validation of RP-HPLC Method and GC MS Analysis of Desloratadine HCL and It’s Degradation Products" is awarded Best Article of Vol 13 issue 21
A Study by Isha Gaurav et al. entitled "Association of ABO Blood Group with Oral Cancer and Precancer – A Case-control Study" is awarded Best Article for Vol 13 issue 20
A Study by Amr Y. Zakaria et al. entitled "Single Nucleotide Polymorphisms of ATP-Binding Cassette Gene(ABCC3 rs4793665) affect High Dose Methotrexate-Induced Nephrotoxicity in Children with Osteosarcoma" is awarded Best Article for Vol 13 issue 19
A Study by Kholis Ernawati et al. entitled "The Utilization of Mobile-Based Information Technology in the Management of Dengue Fever in the Community Year 2019-2020: Systematic Review" is awarded Best Article for Vol 13 issue 18
A Study by Bhat Asifa et al. entitled "Efficacy of Modified Carbapenem Inactivation Method for Carbapenemase Detection and Comparative Evaluation with Polymerase Chain Reaction for the Identification of Carbapenemase Producing Klebsiella pneumonia Isolates" is awarded Best Article for Vol 13 issue 17
A Study by Gupta R. et al. entitled "A Clinical Study of Paediatric Tracheostomy: Our Experience in a Tertiary Care Hospital in North India" is awarded Best Article for Vol 13 issue 16
A Study by Chandran Anand et al. entitled "A Prospective Study on Assessment of Quality of Life of Patients Receiving Sorafenib for Hepatocellular Carcinoma" is awarded Best article for Vol 13 issue 15
A Study by Rosa PS et al. entitled "Emotional State Due to the Covid – 19 Pandemic in People Residing in a Vulnerable Area in North Lima" is awarded Best Article for Vol 13 issue 14
A Study by Suvarna Sunder J et al. entitled "Endodontic Revascularization of Necrotic Permanent Anterior Tooth with Platelet Rich Fibrin, Platelet Rich Plasma, and Blood Clot - A Comparative Study" is awarded Best Article for Vol 13 issue 13
A Study by Mona Isam Eldin Osman et al. entitled "Psychological Impact and Risk Factors of Sexual Abuse on Sudanese Children in Khartoum State" is awarded Best Article for Vol 13 issue 12
A Study by Khaw Ming Sheng & Sathiapriya Ramiah entitled "Web Based Suicide Prevention Application for Patients Suffering from Depression" is awarded Best Article for Vol 13 issue 11
A Study by Purushottam S. G. et al. entitled "Development of Fenofibrate Solid Dispersions for the Plausible Aqueous Solubility Augmentation of this BCS Class-II Drug" is awarded Best article for Vol 13 issue 10
A Study by Kumar S. et al. entitled "A Study on Clinical Spectrum, Laboratory Profile, Complications and Outcome of Pediatric Scrub Typhus Patients Admitted to an Intensive Care Unit from a Tertiary Care Hospital from Eastern India" is awarded Best Article for Vol 13 issue 09
A Study by Mardhiah Kamaruddin et al. entitled "The Pattern of Creatinine Clearance in Gestational and Chronic Hypertension Women from the Third Trimester to 12 Weeks Postpartum" is awarded Best Article for Vol 13 issue 08
A Study by Sarmila G. B. et al. entitled "Study to Compare the Efficacy of Orally Administered Melatonin and Clonidine for Attenuation of Hemodynamic Response During Laryngoscopy and Endotracheal Intubation in Gastrointestinal Surgeries" is awarded Best Article for Vol 13 issue 07
A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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