Potential bioactive phytochemicals,antioxidant properties and anticancer pathways of Nymphaea nouchali

Md.Nazim Uddin,Md.Abdus Samad,Md.Abu Zubair,Md.Zahurul Haque,Kanika Mitra,Tanzir Ahmed Khan,Md.Amir Hossain,Ashikujaman Syed,Aklima Afroze

1Institute of Food Science and Technology,Bangladesh Council of Scientific and Industrial Research,Dhaka 1205,Bangladesh

2School of Basic Medicine and Clinical Pharmacy,China Pharmaceutical University,Nanjing,Jiangsu 211198,China

3Department of Food Technology and Nutritional Science,Mawlana Bhashani Science and Technology University,Santosh,Tangail 1902,Bangladesh

4Center for New Drug Safety Evaluation and Research,China Pharmaceutical University,Jiangsu,Nanjing 211198,China

5School of Pharmacy,Lanzhou University,Lanzhou 730000,China

6School of Pharmacy,China Pharmaceutical University,Nanjing,Jiangsu 211198,China

ABSTRACT

KEYWORDS: Bioactive phytochemicals; Anticancer pathways;Malignancy; Network pharmacology; Nymphaea nouchali

1.Introduction

Nymphaea nouchali (N.nouchali) (Burm.f) is a flowering plant belonging to the family Nymphaeceae.It is a national flower of Bangladesh and Srilanka,commonly known as “Shapla” in Bangladesh.N.nouchali grows abundantly as a mixed population in nearly all shoals and natural water bodies[1].This plant has various bioactivities including anti-inflammatory and diuretic activities[2].In the Ayurveda and Siddha systems of medicines,it is used for the treatment of diabetes,inflammation,liver disorders,urinary disorders,menorrhagia,blennorrhagia,menstruation problem,and used as an aphrodisiac,and a bitter tonic[3,4].Rhizomes and flowers have been widely used for the treatment of kidney problems[3].Flower extract can attenuate melanogenesis by regulation of cAMP/CREB/MAPKs/MITF pathway and proteasomal degradation of tyrosinase[1].Alam et al.revealed that N.nouchali flower extracts had DNA-protecting activities by phosphorylating mitogenactivated protein kinase (MAP kinase) followed by enhancing the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2).These cellular signaling activities attenuate cellular ROS generation and are associated with the protection from cell death[5].The presences of gallic acid,catechin,epicatechin,epigallocatechin,epicatechin gallate,caffeic acid,quercetin,and apigenin were identified in the flower of this plant[5].N.nouchali flower also has antimicrobial activity on human and plant pathogenic bacteria and endophytic fungi[6].Seed extract promotes adipocyte differentiation and glucose consumption by inducing the PPARγ activation,which in turn increases mRNA GLUT-4 expression[7].These previous studies proved the medicinal value of N.nouchali at molecular levels.Plant-derived natural dietary bioactive phytochemicals have potent antioxidants and cancer chemopreventive agents[8].Dietary phytochemicals are often used as anticancer agents against breast cancer,skin cancer,diverse types of thyroid cancers,prostate cancer,and gastroenterological cancer,as well as play a role in modulating coding and non-coding genes in cancers[9].N.nouchali tubers are eaten usually boiled or roasted.A novel Ca2+-dependent lectin was isolated from the N.nouchali tuber and exhibited antiproliferative properties[10].Additionally,the tuber and root of N.nouchali were used by the folk medicine practitioners in three districts of Bangladesh for the prevention and management of malignancy in cancer[11].However,to the best of our knowledge,there have been no investigations to date that identify the subset of anticancer pathways associated with the bioactive phytochemicals of N.nouchali tuber extract.Therefore,we designed the present study to identify the potential bioactive phytochemicals,to evaluate in vitro antioxidant properties,and to investigate the predicted genes interacting with bioactive phytochemicals and the association of these predicted genes with cancer pathways.In addition,we investigated the hub genes,which are regulated by the bioactive phytochemicals of N.nouchali.

2.Materials and methods

2.1.Collection and preparation of methanolic crude extract of N.nouchali

The N.nouchali tubers were collected from different canals and ponds in Bangladesh.Collected tubers were thoroughly washed with fresh water,dried under shade at room temperature for 2 d.Then the skin of the tubers was removed and the tubers were cut into thin pieces.The pieces were sun-dried and powdered in a mechanical blender.About 100 g of dry powders were macerated in 500 mL methanol for 3 d in a flat bottomed container with occasional shaking,stirring,homogenization,and sonication[12].Then it was filtered through a filter paper and the supernatant was collected.The extraction was repeated three times.The methanol was evaporated under reduced pressure below 50 ℃ through the rotatory evaporator(RE 200 Sterling,UK).The concentrated N.nouchali tuber extract was stored at 4 ℃ until further use.

2.2.Quantification of bioactive phytochemicals in methanolic extract of N.nouchali tuber

The amount of total phenolic and total tannin content was determined following the established method with some modifications by using Folin-Ciocalteu reagent[13].The content of total flavonoids and proanthocyanidins was determined spectrophotometrically using a standard curve of catechin[14].Ascorbic acid was assessed based on the established procedure[15].Methanolic extract of N.nouchali tuber was then subjected to the high-performance liquid chromatography-diode array detector (HPLC-DAD) analysis.The composition of bioactive phytochemicals was determined by HPLC[14,16].Chromatographic analyses were carried out on a Thermo Scientific Dionex ultimate 3000 rapid separation LC (RSLC) systems (Thermo Fisher Scientific Inc.,MA,USA),coupled to a quaternary rapid separation pump(LPG-3400RS),Ultimate 3000RS autosampler (WPS-3000) and rapid separation diode array detector (DAD-3000RS).Bioactive phytochemicals were separated on an Acclaim®C18 (4.6 mm × 250 mm × 5 µm) column (Dionix,USA),which was controlled at 30 ℃using a temperature-controlled column compartment (TCC-3000).For the preparation of calibration curve,a standard stock solution was prepared in methanol containing all bioactive phytochemicals.Data acquisition,peak integration,and calibrations were performed with Dionix Chromeleon software (Version 6.80 RS 10).At least three determinations were conducted for every analysis.

2.3.In vitro antioxidant activity

Total antioxidant activity was determined by using the phosphomolybdenum blue and FRAP reagent[14].2,2-diphenyl-1-picrylhydrazyl (DPPH),2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),and N,N-dimethyl-p-phenylenediamine(DMPD) were used to evaluate the in vitro radical scavenging activity[14].Standard ascorbic acid was used in DPPH scavenging assay and gallic acid was used as a standard in ABTS and DMPD scavenging assays.The reducing power was determined by using ascorbic acid as a standard.The chelation of ferrous ions and superoxide radical scavenging capacity were also assessed according to our previous work[14,17].Based on the screening results of the triplicate measurement of the extract,the inhibition concentration (IC50) value was determined from extrapolating the graph of scavenging activity versus the concentration of extract(using linear regression analysis),which is defined as the amount of antioxidant necessary to reduce the initial radical concentration by 50%.EC50value was determined from extrapolating the graph of the concentration of extract or standard versus absorbance at a specific nanometer (nm) using linear regression analysis.EC50is the concentration of extract or standard to obtain absorbance=0.50[14].

2.4.Bioinformatics analysis

2.4.1.Target gene prediction for bioactive phytochemicals

On the basis of network pharmacology-based prediction,STITCH 5 (http://stitch.embl.de/) was used to identify target genes related to bioactive phytochemicals[18].It calculates a score for each pair of protein-chemical interactions.Chemical names of bioactive compounds (rutin,catechin,myricetin,ellagic acid,gallic acid,vanillic acid,rosmarinic acid,p-coumaric acid,quercetin,and ascorbic acid) were input into STITCH 5 singly to match their potential targets,with the organism selected as “Homo sapiens” and minimum required interaction score being ≥ 0.4.We predicted 20 genes with top confidence score for each bioactive compound by chromatographic and spectrophotometric analysis.The compound targets without a relationship with the compound-proteins interactions were not considered for further analysis.According to the results,only rosmarinic acid was not detected for any target genes.Usually,interactions with score ≥ 0.4 are considered as medium confidence.

2.4.2.Identification of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway by enrichment analyses of the predicted genes

We performed a gene-set enrichment analysis of the predicted genes[19].The KEGG[20]pathways significantly associated with the predicted genes were identified.The false discovery rate (FDR) ＜0.05,calculated by the Benjamini and Hochberg method[21],was considered as significant.

2.4.3.Construction of protein-protein interaction (PPI)network of the predicted genes

We constructed a PPI network of the predicted genes by search tool for the retrieval of interacting genes (STRING) database(https://string-db.org/cgi/input.pl; STRING-DB v11.0)[22].The hub genes (with no less than ten edges connected to other nodes) in the PPI network were identified using the node explorer module of NetworkAnalyst software[23].

3.Results

3.1.Bioactive phytochemicals in the methanolic extract of N.nouchali tuber

The yield of methanolic extract of N.nouchali tuber was 5.49%(w/w).The content of total phenolics [(353.66±2.98) mg/g of the extract]was higher than total flavonoids [(102.86±14.13) mg/g of the extract],total tannins [(78.14±21.28) mg/g of the extract],and total proanthocyanidins [(4.42±2.67) mg/g of the extract].The extract contained a moderate amount of ascorbic acid [(27.57±0.12)mg/100 g sun-dried tuber powder].Furthermore,we identified bioactive phytochemicals using a HPLC system (Figure 1) and the results showed the presence of rutin (39.44 mg),catechin (39.20 mg),myricetin (30.77 mg),ellagic acid (11.05 mg),gallic acid (3.67 mg),vanillic acid (0.75 mg),rosmarinic acid (4.81 mg),p-coumaric acid (3.35 mg),and quercetin (0.90 mg) in 1 g of dry methanolic extract of N.nouchali tuber.

Figure 1.HPLC-DAD chromatogram of the methanolic extract of Nymphaea nouchali tuber.1: gallic acid,2: (+)-catechin,3: vanillic acid,4: p-coumaric acid,5: ellagic acid,6: rutin,7: rosmarinic acid,8: myricetin,and 9:quercetin.

3.2.In-vitro antioxidant activities of methanolic extract of N.nouchali tuber

By using phosphomolybdenum blue,the total antioxidant capacity in the methanolic extract of N.nouchali tuber was determined using the following linear regression equation: y=0.010x-0.090,R2=0.975,where y is absorbance and x is the ascorbic acid concentration in microgram.The total antioxidant capacity was (159.78±23.11) mg ascorbic acid equivalent in 1 g of the methanolic extract.By using FRAP reagents,the total antioxidant capacity was (763.58±36.88)mg ascorbic acid equivalent in 1 g of the methanolic extract (Linear regression equation of standard ascorbic acid: y=0.041x-0.072,R2=0.998).

We used DPPH,ABTS,and DMPD assays to screen free radical scavenging capacity of the extract.The IC50value of standard ascorbic acid and the N.nouchali tuber extract was 13.77 µg/mL and 26.44 µg/mL,respectively in DPPH assay.For the extract and the standard gallic acid in ABTS assay,the IC50value was 25.14 µg/mL and 8.86 µg/mL,respectively.In DMPD assay,the value was 49.32µg/mL and 22.86 µg/mL,respectively.

Moreover,reducing power,superoxide radical scavenging capacity,and chelation of ferrous ions of the methanolic extract were determined.Ascorbic acid and the extract showed the EC50of 44.42µg/mL and 164.50 µg/mL,respectively in reducing power assay,indicating that bioactive phytochemicals of the extract are election donors and can reduce the oxidized intermediates.Superoxide was generated by alkaline DMSO method and the IC50value of standard gallic acid and the methanol extract of the tuber was 75.90 µg/mL and 677.50 µg/mL,respectively.The assay for chelation capability of ferrous ion showed the IC50value of standard ethylene diamine tetraacetic acid and the extract was 30.65 µg/mL and 1 949.69 µg/mL,respectively,which indicates that the extract has extremely poor chelation capacity of ferrous ion.These results clearly indicated that the bioactive phytochemicals responsible for the antioxidant potentials might be present in the methanolic extract of N.nouchali tuber.

3.3.Network-based target prediction for bioactive phytochemicals

A drug-target network was constructed to further examine the potential mechanisms of action of the bioactive phytochemicals.The freely available STITCH software (http://stitch.embl.de/)was used to construct the drug-target network and to provide genetic identification in humans.We selected 20 genes/protein for each bioactive phytochemical identified by chromatographic analysis.Altogether,130 unique target genes were predicted by all the compounds (ascorbic acid,catechin,myricetin,ellagic acid,gallic acid,vanillic acid,p-coumaric acid,quercetin,and rutin).The network-based target predicted genes are summarised in Supplementary Table 1.

3.4.Enrichment of cancer pathways of the predicted genes

Enriched KEGG pathways of the predicted 130 unique genes were analyzed.These pathways were mainly involved in cellular development,immune regulation,metabolism,and cancer(Supplementary Table 2).-Log10(FDR) values of some significantly(FDR＜0.05) enriched cancer-associated pathways are shown in Figure 2.Some of significant cancer-associated pathways(FDR＜0.05) were as follows: pathways in cancer (MAPK3,PIK3CG,AKT1,PRKCA,JUN,CHUK,CXCL8,IL6,CASP3,PTGS2,SLC2A1,ARNT,EGFR,BMP2,MMP2,MMP9,NOS2,and FGF2),renal cell carcinoma (MAPK3,PIK3CG,AKT1,JUN,SLC2A1,and ARNT),bladder cancer (MAPK3,MMP2,EGFR,MMP9,and CXCL8),non-small cell lung cancer (MAPK3,PIK3CG,AKT1,PRKCA,and EGFR),acute myeloid leukemia (MAPK3,PIK3CG,AKT1,CHUK,and PIM1),colorectal cancer (MAPK3,PIK3CG,AKT1,CASP3,and JUN),glioma (MAPK3,PIK3CG,AKT1,PRKCA,and EGFR),pancreatic cancer (MAPK3,PIK3CG,AKT1,CHUK,and EGFR),melanoma (MAPK3,PIK3CG,AKT1,FGF2,and EGFR),small cell lung cancer (PIK3CG,AKT1,NOS2,CHUK,and PTGS2),prostate cancer (MAPK3,PIK3CG,AKT1,CHUK,and EGFR),endometrial cancer (MAPK3,PIK3CG,AKT1,and EGFR),and chronic myeloid leukemia (MAPK3,PIK3CG,AKT1,and CHUK).

3.5.Identification of hub genes associated with pathways in cancer

Using STRING database and NetworkAnalyst software,a total of 130 predicted genes were mapped into the PPI network,including 128 nodes and 841 edges with the PPI enrichment P-value less than 1.0 × 10−16.Interestingly,we found that 117 genes were involved in the PPI network and 65 genes (hub genes) had PPI connectivity degree not less than 10 (Figure 3 and Supplementary Table 3).In addition,we identified top ten hub genes (IL6,AKT1,EGFR,CAT,JUN,PTGS2,MAPK3,CASP3,CXCL8,and NOS3) with a higher degree of connectivity,which are associated with pathways in cancer in this study,and eight of them interacted with bioactive phytochemicals of the methanolic extract of N.nouchali tuber(Figure 4 and Table 1).These hub genes interacted with one or more bioactive phytochemicals.For example,AKT1 interacted with gallic acid,myricetin,and quercetin; CASP3 with gallic acid,rutin,quercetin,and myricetin.Based on the hub genes and the bioactive phytochemicals,we established a gene-bioactive phytochemicals regulatory network that may contribute to the treatment of cancer.This network included the interactions of IL6-catechin,AKT1-gallic acid/myricetin/quercetin,EGFR/MAPK3/CXCL8-rutin,JUN-gallic acid,PTGS2-catechin/p-coumaric acid/quercetin,and CASP3-gallic acid/rutin/quercetin/myricetin.

Figure 2.Some of significantly enriched cancer-associated KEGG pathways in human.FDR: false discovery rate.

Table 1.The top ten hub genes with a higher degree of connectivity which are targeted by bioactive phytochemicals from the methanolic extract of Nymphaea nouchali tuber.

Figure 3.Protein-protein interaction network of the 130 predicted genes.117 genes were involved in the protein-protein interaction network.

Figure 4.Hub genes (CXCL8,EGFR,MAPK3,JUN,CASP3,AKT1,PTGS2,and IL6) interacted with bioactive phytochemicals of Nymphaea nouchali tuber extract (NNTE) and associated with cancer pathways.

4.Discussion

Phenolics have remarkable bioactivities including anticancer,anti-inflammatory,antibacterial,anti-diabetes,anti-cardiovascular,anti-neurodegenerative,anti-analgesic,anti-allergic,and anti-Alzheimer’s properties[24].Phenolics also stimulate the expression of tumor-suppressing proteins such as p53,phosphatase and tensin homolog (PTEN),p21,and p27[25].The biological and pharmacological effects of flavonoids include antioxidant,antiinflammatory,cardioprotective,hepatoprotective,antimicrobial,and anticancer activities[26].Proanthocyanidins and tannins have various pharmacological effects,including antioxidant and free radical scavenging activity,antimicrobial,anti-cancer,anti-nutritional,and cardioprotective properties[27].Our study showed the presence of bioactive phytochemicals in methanolic extract of N.nouchali tuber.Natural antioxidants exhibit a wide range of biological effects,such as anti-inflammatory,antibacterial,antiviral,anti-aging,and anticancer[28].Therefore,our results suggested that the extract of N.nouchali tuber might be used as an effective and safe source of natural antioxidants against various diseases,including cancer.

Network medicine offers a platform to systematically explore the molecular complexity of a particular disease,which helps identify modules and pathways of diseases as well as the molecular relationships among apparently distinct phenotypes[29].Integrating network biology and pharmacology is a paradigm for drug discovery[30].Phytochemicals activate pathways and thereby enhance the ability of cells to resist injury and disease[31].Dietary phytochemicals demonstrate strong anticancer effects and affect several cancer-related pathways[32].Dietary bioactive phytochemicals have gained tremendous attention because of their ability to inhibit multiple signaling pathways as a promising approach to prevent and treat cancers[33].Our findings indicate that bioactive phytochemicals in the methanolic extract of N.nouchali tuber are associated with anticancer pathways,and these bioactive phytochemicals may be better therapeutic agents for treating cancers.PPI network plays an important role in molecular processes,and abnormal PPI is the basis of many pathological conditions,including tumors[34].The IL-6/JAK/STAT3 pathway is aberrantly hyperactivated in many types of cancer[35].Another hub gene PTGS2 is upregulated during both inflammation and cancer[36].IL6 and PTGS2 both interact with catechin,and it was stated that catechin intake may ensure a protective epigenetic status against the adverse effects through the interaction with IL6[37].An important antioxidant enzyme catalase(CAT) is another hub gene with 46 connectivity degree and is associated with genetic,epigenetic,and posttranscriptional processes.Abnormal expression levels of CAT have been reported in cancer tissues[38].Gallic acid significantly increased the activity of CAT in rat model.A recent study demonstrated the link between aberrant cell cycle progression and AKT hyperactivation in cancer[39].Gallic acid inhibited the activities of AKT as well as migration and invasion in human osteosarcoma[40].Quercetin facilitates cell death and chemosensitivity through RAGE/PI3K/AKT/mTOR axis in human pancreatic cancer cells[41].A proto-oncogene JUN is a molecular target for improving cancer therapy[42].CASP3 is used as a marker for efficacy of cancer therapy and this gene may not only increase the sensitivity of cancer cells to chemotherapy and radiotherapy,but also inhibit cancer cell invasion and metastases[43].Gallic acid,rutin,tannic acid,and quercetin significantly inhibited cell proliferation of human prostate cancer by increasing the caspase-3 activity[44].Gallic acid also upregulated the expression of CASP3 that induced apoptosis[45].Aberrant expression of MAPK3 is associated with invasion,metastasis,and drug resistance of multiple tumor cells[46].EGFR is involved in the pathogenesis and progression of different carcinoma types[47].CXCL8 is the most studied chemokine in human malignancies and displays multifaceted pro-tumorigenic effects,including tumor cell growth,metastatization,and angiogenesis,contributing to the progression of several cancers[48].Polyphenols downregulated gene expression of pro-inflammatory cytokines/enzymes and differentially modulated the inflammatory response of human keratinocytes through distinct signal transduction pathways,including EGFR-MAPK pathways[49].Our results reveal that three hub genes (MAPK3,EGFR,and CXCL8) interacted with rutin.The above findings suggested that these hub genes might play a crucial role in various types of cancers and interact with bioactive compounds of the N.nouchali tuber extract.

5.Conclusion

The discovery of bioactive phytochemicals and their predicted target genes will be of great importance in various treatments against chronic diseases including various cancers.The results strongly indicate that this medicinal plant can serve as a very useful source of antioxidant and anticancer components.The network pharmacology contributes to understanding the complex interactions between bioactive phytochemicals and anticancer pathways from a network perspective.Consequently,network pharmacology provides a novel approach to promote drug discovery in a precise manner from this plant.Furthermore,clinical experiments are required to characterize the potent molecule from the pharmaceutical point of view in future studies.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Funding

This work was carried out under the research and development (R and D) project of the Bangladesh Council of Scientific and Industrial Research.

Authors’ contributions

MNU performed data analyses,conceived the research,designed analysis strategies,and wrote the manuscript.MZH and MAZ conceived the research and designed analysis strategies.MAS and TAK performed data analyses.MAH,AS,and AA drew the figures and helped write the manuscript.KM helped to revise the manuscript.All the authors read and approved the final manuscript.