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ORIGINAL ARTICLE
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Biochemical and molecular anticancer approaches for Boerhaavia diffusa root extracts in oral cancer


1 Department of Chemistry and Biosciences, Srinivasa Ramanujan Center, SASTRA Deemed University, Kumbakonam, Tamil Nadu, India
2 Department of Biochemistry, Cancer Biology Unit, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India
3 Department of Histopathology, Christian Medical College and Hospital, Vellore, Tamil Nadu, India
4 Department of Chemistry and Biosciences, Srinivasa Ramanujan Center, SASTRA Deemed University, Kumbakonam; Department of Technology Dissemination, Indian Institute of Food Processing Technology, Thanjavur, Tamil Nadu, India

Date of Submission07-Jul-2020
Date of Decision29-Aug-2020
Date of Acceptance07-Oct-2020
Date of Web Publication24-Jul-2021

Correspondence Address:
Baskaran Nagarethinam,
Department of Technology Dissemination, Indian Institute of Food Processing Technology, Pudukottai Road, Thanjavur - 613 005, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_932_20

 > Abstract 


Background: Boerhaavia diffusa is a medicinal herb with anti-inflammatory, antiproliferative, anticancer, and immunomodulatory properties, found across India.
Aim and Objectives: The present study is designed to investigate the therapeutic potential for B. diffusa root extracts in oral cancer cell line.
Materials and Methods: The aqueous and methanolic extracts of B. diffusa were prepared using Soxhlet apparatus. In order to determine the phytochemical constituents of B. diffusa, the extracts were subjected to gas chromatography-mass spectrometry analysis. The antioxidant potential of B. diffusa extracts was assessed by 2,2-Diphenyl-picrylhydrazyl, ferric ion-reducing antioxidant power, catalase and peroxidase assays. The effective concentration of B. diffusa root on cell viability was analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The ability of B. diffusa root extracts to modify the cell-cycle phases was performed by FACS analysis. The apoptotic inducing potential of B. diffusa in oral cancer cells was confirmed by acridine orange-ethidium bromide and 4′,6-diamidino-2-phenylindole staining. The protein profile of apoptotic processes was validated by the Western blot analysis; docking studies were also performed.
Results: We observed that antioxidant activity was higher in B. diffusa methanolic extract compared with aqueous extract. The results showed that the methanolic and aqueous extracts of B. diffusa exhibited significant cytotoxic effect with IC50 value of 36 μg/ml and 30 μg/ml, respectively. The apoptotic DNA fragmentation and the apoptotic inducing potential in KB oral cancer cell line were higher for the methanolic extract compared with the aqueous extract. These results were also confirmed by in-silico analysis.
Conclusion: The results indicate that extracts obtained from the roots of B. diffusa inhibit the progression of oral cancer. These compounds of pharmacological importance can be either used alone or in combination with other drugs to treat oral cancer.

Keywords: Anticancer, antioxidant, Boerhaavia diffusa, KB cells, oral cancer



How to cite this URL:
Gunaseelan D, Ali MS, Albert A, Prabhakaran R, Beno DL, Nagarethinam B. Biochemical and molecular anticancer approaches for Boerhaavia diffusa root extracts in oral cancer. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322274




 > Introduction Top


Oral cancer or mouth cancer is the sixth most common cancer globally and third most prevalent cancer in India. Oral squamous cell carcinoma (OSCC) is the most common in both Asian (80.23%) and non-Asian countries (Nigeria, Libya) (73.46%).[1] The World Health Organization estimated that 657,000 new cases and >330,000 deaths occur due to oral and pharyngeal cancer each year.[2] In India, around 1, 20,000 new cases and 72,000 deaths occurs every year[3] accounting for around 30% of all cancers. It usually affects both genders and is detected late; OSCC being accompanied with poor prognosis and high-cost therapies result in higher mortality. Oral cancer includes cancerous growth seen in the lips, buccal mucosa, teeth, gingiva, tongue, the floor of mouth, gums, soft and hard palate, and pharynx. Approximately, 90% of cancers in the oral cavity are squamous cell carcinomas, and the remaining can be melanomas, salivary gland malignancies, lymphomas, sarcomas, and metastatic cancer. Tobacco use and alcohol consumption are two of the most common risk factors for oral cancer.

Chemoprevention is the prevention of initiation, promotion, and progression of carcinogenesis to cancer. Retinoids are the most extensively studied agents for the chemoprevention of oral cancer.[4] Dietary factors play a key role for the prevention of human diseases including cancer. Many natural compounds from green and yellow-colored vegetables, fruits, and spices act as chemopreventive agents. For example, polyphenols (PPs) from green tea possess powerful antimicrobial and antioxidant activity that is capable of scavenging reactive oxygen, nitrogen, superoxide anions, and metal ions. By having direct contact with tissues at the biologically active aglycones on the surface of the epithelial cells, PPs inhibit the proliferation of oral cancer cells.[5] Curcumin, a hydrophobic PP, isolated from the rhizomes of the plant Curcuma longa L possess cytotoxic, anti-inflammatory, antioxidant, immunomodulatory, anti-angiogenic, and apoptotic properties.[6],[7],[8] There are also many other natural agents such as tomatoes, red carrots, guava, watermelons, papayas, celery, green pepper, thyme, broccoli, cabbage, spinach, olive oil, peppermint, rosemary, and oregano with chemopreventive activity.[9] Understanding the process of molecular carcinogenesis has made it possible for the identification of natural chemopreventive agents to target the molecular mechanisms of carcinogenesis.

Boerhaavia diffusa (B. diffusa) is a medicinal herb, commonly called punarnava in the Indian medicine system. It is a perennial weed belonging to the family of Nyctacinaceae. It is found in tropical and subtropical regions such as India, Brazil, Africa, Australia, China, Egypt, Pakistan, USA, and Sri Lanka. B. diffusa has a high nutritional content B. diffusa and is also used as a green vegetable in India. The plant harbors 15 amino acids (6 essential) and fatty acids such as isopalmitate acetate, behenic acid, and arachidic acid. In addition, Vitamins C, B2 and B3 and minerals like calcium are also present in abundance.[10] In vitro studies have shown that B. diffusa exhibit anti-carcinogenic effects against human breast cancer cells. It has also been reported to inhibit pro-inflammatory mediators such as (secretory phospholipase A2) in experimental inflammatory systems.[11] B. diffusa has diverse pharmacological properties including immunomodulatory, anti-cell proliferative, antioxidant, anti-tumor, anti-bacterial, and anti-fungal activity.[12],[13],[14] Previous studies have demonstrated that the extracts from B. diffusa plants have it's the ability to scavenge free radicals.[15] However, the use of B. diffusa root extracts against oral cancer remains elusive. To the best our knowledge, there are no scientific studies that document the chemopreventive potential of B. diffusa root extracts against KB oral cancer cell line. The present study was therefore designed to explore the molecular anticancerous mechanism of B. diffusa root extracts in oral cancer cell line. The aqueous and methanolic extracts of B. diffusa were compared in terms of composition, antioxidant potential, and chemopreventive activity.


 > Materials and Methods Top


Chemicals

Methanol, Dulbecco's modified eaglesmedium (DMEM), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), acridine orange (AO), ethidium bromide (EtBr), dimethyl sulfoxide (DMSO), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide, 2,2-Diphenyl-picrylhydrazyl (DPPH) were purchased from HiMedia chemicals, India. All other chemicals and solvents were of analytical grade and purchased from the usual sources.

Plant collection

B. diffusa roots were collected from SRC, SASTRA campus, Kumbakonam, Tamil Nadu, India. The plant material was identified and authenticated by Dr. N. Ravichandran, Center for Advanced Research in Indian System of Medicine, SASTRA Deemed University, Tirumalaisamuthiram, Thanjavur, Tamil Nadu, India.

Preparation of plant extracts

The roots were washed with distilled water and cut into small pieces, shade dried at room temperature for 2–3 days, ground into powder and used for extraction. The powdered root of B. diffusa was packed in a unit of Soxhlet apparatus and extracted with methanol (50 g of sample + 500 ml of methanol) and water (50 g of sample + 500 ml of water) at 64.7°C and 100°C, respectively. When the volume of the solvent became sufficient, the concentrated liquid was collected and dried. The extract was stored at 4°C for further analysis.[16]

Preparation of sample solution

B. diffusa extracts were dissolved in 0.2% DMSO. The stock solution (1 mg/ml) was diluted with DMEM medium to produce a working concentration of 10, 25, and 50 μg/ml.

Cell culture

KB cell lines were purchased from the National Center for Cell Sciences, Pune, India. The cancer cells were maintained in DMEM supplemented with 2 mM l-glutamine and balanced salt solution adjusted to contain 1.5 g/L Na2CO3, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 1.5 g/L glucose, 10 mM (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid), and 10% fetal bovine serum (GIBCO, USA). Concentrations of penicillin and streptomycin (100 IU/100 μg) were adjusted to 1 mL/L. The cells were maintained at 37°C with 5% CO2 in a humidified CO2 incubator.

Phytochemical, antioxidant, and apoptotic activity

The B. diffusa extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). The antioxidant potential of the extracts was determined by ferric ion-reducing antioxidant power (FRAP), DPPH free radical scavenging, catalase (CAT) and peroxidase (POD) assay. The apoptotic property of the extract was studied through MTT assay, AO/EB staining, DAPI staining, cell cycle analysis, and Western blotting analysis [Table 1].[17],[18],[19],[20],[21],[22],[23],[24],[25]
Table 1: Evaluation of Boerhavia diffusa in phytochemical, antioxidants and apoptotic protein analysis

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In-silico analysis

Three dimensional structures of Bcl-2 and BAD proteins were downloaded from PDB database (https://www.rcsb.org/structure/1G5J) (PDB ID FOR BCl2-1G5M and BAD-1G5J). The 2D structure of 9,12-Octadecadienoic acid (z-z) was obtained from PUBCHEM database in SDF format and converted to PDB format using online molecular converter tool (http://cactus.nci. nih.gov/translate). The docking analysis with the 3D structure of Bcl-2 and BAD protein was carried out by Patch dock. Patch dock was used to find the binding efficiency of molecules. The structure was visualized, and the docked complexes were analyzed using PyMOL.[26]

Statistical analysis

All experiments were repeated at least thrice. The statistical software SPSS version 17.0 (SPSS Inc. 233, South Walker Drive, 11th Floor, Chicago, IL) was used for the analysis. P < 0.05 was considered statistically significant.


 > Results Top


Preliminary qualitative phytochemical analysis of plant extracts

The preliminary phytochemical screening of B. diffusa root extracts showed the presence of major phytoconstituents such as alkaloids, anthraquinones, flavonoids, glycosides, reducing sugar, saponins, steroids, tannins, and terpenoids [Table 2]. The methanolic extract of B. diffusa (BDME) exhibited the increased levels of phytoconstituents than the aqueous extract of B. diffusa (BDAE). The methanolic extract comprised phytochemicals such as alkaloids, anthraquinones, flavonoids, saponins, and tannins. The aqueous extract showed the presence of alkaloids, anthraquinones, flavonoids, reducing sugar, tannins, and terpenoids.
Table 2: The qualitative phytochemical analysis of Boerhaavia diffusa in aqueous and methanol extracts

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Gas chromatography-mass spectrometry

The presence of bioactive compounds was identified in B. diffusa root by GC-MS analysis. The active principles with their retention time, molecular formula, molecular weight (MW), and concentration (%) are presented in [Table 3]. The compounds present are thiophene, 2-ethyltetrahydro-(4.69), tetradecanoic acid (0.44), hexadecanoic acid, methyl ester (0.25), n-hexadecanoic acid (9.08), methyl 10-trans 12-cis-octadecadienoate (2.87), 11-octadecenoic acid, methyl ester 3.17, 9, 12-octadecadienoic acid [Z, Z]-(78.5). Thus, the bioactive compounds analyzed by GC-MS give a comprehensible presentation of the pharmaceutical and therapeutic value of the plant. [Figure 1] shows GC-profile, MS-profile, and structures of 9, 12-octadecadienoic acid [Z, Z]-.
Table 3: Bioactive compounds identified from methanolic extract of Boerhaavia diffusa by gas chromatography-mass spectrometry

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Figure 1: The gas chromatography-mass spectrometry analysis of methanolic extract of Boerhaavia diffusa: (a) Bioactive compound-9, 12-Octadecadienoic acid [Z, Z]-.; (b) gas chromatography-profile of Boerhaavia diffusa (c) mass spectrometry–profile of Boerhaavia diffusa

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Extracts of Boerhaavia diffusa exhibited ferric ion-reducing ability

The reducing effect of methanolic and aqueous root extracts of B. diffusa at different concentrations (20–100 μg/ml) were determined using FRAP assay. The methanolic extract (19.43%) of B. diffusa exhibited higher reducing activity of free radicals compared to the aqueous extract [16.51%; [Figure 2]a for details]. The assay measured the antioxidant potential through the conversion of ferric ion to ferrous ion by antioxidants present in the plant extract. The reducing properties are generally associated with the presence of reductones which have been shown to exert antioxidant action by breaking the free radical chain by means of donating a hydrogen atom.
Figure 2: Antioxidant assays: (a) The antioxidant activity of Boerhaavia diffusa extracts by ferric ion reducing antioxidant power reducing assay. (b) The antioxidant activity of Boerhaavia diffusa extracts by 2,2-Diphenyl-picrylhydrazyl free radical scavenging assay. (c) The antioxidant activity of Boerhaavia diffusa extracts by Catalase assay. (d) The antioxidant activity of Boerhaavia diffusa extracts peroxidase assay. Ascorbic acid was used as a standard and the values are represented as mean ± standard deviation

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Free radical scavenging property of Boerhaavia diffusa extract

The free radical-scavenging effect of methanolic and aqueous root extract of B. diffusa at different concentrations (20–100 μg/ml) was determined using 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH). The results are expressed in the percentage of scavenging activity. Our results showed that methanolic extracts had maximum DPPH radical scavenging activity compared with the BDAE [63.33% vs. 55.56%; [Figure 2]b]. Similarly, in comparison to the aqueous extract, the BDME showed higher CAT activity [27.54% vs. 15.04%; [Figure 2]c]. POD assay revealed that the methanolic extract (21.88%) of B. diffusa showed higher scavenging activity compared to the aqueous extract (10.16%) [Figure 2]d.

Cytotoxic effect of Boerhaavia diffusa on KB cells by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay

We analyzed the cell response of human oral cancer cells when exposed to extracts by using MTT assay. [Figure 3] (3.1 and 3.2) shows the in vitro cytotoxicity activity of the extracts (10, 20, 30, 40 and 50 μg/ml concentrations) against selected cancer cells. The experimental results demonstrated that the complex inhibited cell proliferation in a dose-dependent manner. The IC50 values of aqueous and methanol extracts against oral cancer cells were 36 and 30 μg/ml, respectively. It can be noticed from the results that both extracts significantly inhibited the proliferation of selected human cancer cells.
Figure 3: The cytotoxic effect Boerhaavia diffusa extract against KB oral cancer cells. (a): The effect of aqueous extract of Boerhaavia diffusa at different concentrations. (b) The effect of methanolic extract of Boerhaavia diffusa at different concentrations. Values are represented as mean ± standard deviation of three experiments

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Cell morphology analysis

The morphological changes of selected cancer cells in the absence and presence of extracts at various concentrations (10, 25, and 50 μg/ml) are shown in [Figure 4] (4.1 and 4.2). Control cells did not show any remarkable changes in their morphology. However, in the presence of either extract, the cells showed higher shrinkage, membrane blebbing, and nonviable in a dose-dependent manner.
Figure 4: (4.1 and 4.2) Morphological appearance of KB cells after the treatment of aqueous and methanolic extracts of Boerhaavia diffusa (after 24 h), respectively: (a) Untreated KB oral cancer cells (control), (b) cancer cells (KB) + Boerhaavia diffusa (10 μg/ml), (c) cancer cells (KB) + Boerhaavia diffusa (25 μg/ml), (d) cancer cells (KB) + Boerhaavia diffusa (50 μg/ml)

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The effect of Boerhaavia diffusa on apoptotic morphological changes in KB cells

In order to study the effect of the apoptogenic activity of the extracts, cells were stained with AO/EtBr. Fluorescence microscopy images of KB cells in the presence and absence of extracts are depicted in [Figure 5]a and [Figure 5]b. Untreated KB cells did not show experience significant adverse effects compared to the treated cell group. The orange/red fluorecence in cells treated with the extracts were indicative of apoptosis and nuclear condensation. In order to further validate the nuclear fragmentation of cancer cells, DAPI staining method was performed. It was seen in [Figure 6] (6.1 and 6.2) that untreated cells did not show any significant changes, whereas KB cells treated with either plant extract showed bright spots indicative of condensed chromatins and nuclear fragmentations. The results from fluorescence microscopy analysis were suggestive of the potent therapeutic agents.
Figure 5: (5.1 and 5.2) Apoptotic effect of aqueous and methanolic extracts of Boerhaavia diffusa on KB oral cancer cells by acridine orange/ethidium bromide staining. (a) Untreated KB oral cancer cells (control), (b) cancer cells (KB) + Boerhaavia diffusa (10 μg/ml), (c) cancer cells (KB) + Boerhaavia diffusa (25 μg/ml), (d) cancer cells (KB) + Boerhaavia diffusa (50 μg/ml)

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Figure 6: (6.1 and 6.2) Fluorescence microscopic images of KB oral cancer cells after treatment with aqueous and methanolic extracts of Boerhaavia diffusa by 4',6-diamidino-2-phenylindole staining, respectively. (a) Untreated KB oral cancer cells (control), (b) cancer cells (KB) + Boerhaavia diffusa (10 μg/ml), (c) cancer cells (KB) + Boerhaavia diffusa (25 μg/ml), (d) cancer cells (KB) + Boerhaavia diffusa (50 μg/ml)

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Cell cycle analysis

Both extracts (aqueous and methanol) were able to stimulate the cell cycle arrest at various phases ([Figure 7] (7.1 and 7.2). In the untreated control, there was an accumulation of cells in the G0/G1-phase. The percentage of cells in the G0/G1-phase significantly increased on treatment with the extracts. Moreover, compared to the control, treatment with the extract elevated the DNA duplications. The results indicated that the extract could be predominantly derived from inducing cell cycle phase activation from arrest mainly in DNA duplication phase.
Figure 7: (7.1 and 7.2) Cell cycle changes in KB cells after treatment with aqueous and methanolic extracts of Boerhaavia diffusa by FACS using propidium iodide respectively. (a) Untreated KB oral cancer cells (control), (b) cancer cells (KB) + Boerhaavia diffusa (10 μg/ml), (c) cancer cells (KB) + Boerhaavia diffusa (25 μg/ml), (d) cancer cells (KB) + Boerhaavia diffusa (50 μg/ml)

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Immunoblot

Expression profile of Bad and Bcl-2 proteins were analyzed in KB cells treated with the extracts using western blotting [[Figure 8] (8.1 and 8.2)]. Treatment of KB cells with the extract significantly up-regulated Bad protein and downregulated Bcl-2 protein expression compared with untreated control cells. Thus, our results indicate that both extracts exhibit strong apoptotic potential particularly in cancer cells.
Figure 8: (8.1 and 8.2 a): Immunodetection of Bad and Bcl-2 protein expression by the Western blotting after treatment with aqueous and methanolic extracts of Boerhaavia diffusa against KB cells, respectively, (lane 1: Untreated KB oral cancer cells [control], lane 2: Cancer cells [KB] + Boerhaavia diffusa [10 μg/ml] lane 3: Cancer cells [KB] + Boerhaavia diffusa [25 μg/ml] lane 4: Cancer cells [KB] + Boerhaavia diffusa [50 μg/ml]. β-actin [42 kDa] was used as an internal control.(b): Relative gene expression of Bad and Bcl-2

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Docking studies

The compound 9,12 Octadecadienoic acid [Z, Z]-interacts with anti-apoptotic and proapoptotic protein [Figure 9]. The interaction of Bcl-2 protein bound with 9,12 octadecadienoic acid [Z, Z]-was higher than Bad protein [Table 4]. These interaction studies indicate that the compound [9,12 Octadecadienoic acid [Z, Z]-] present in the B. diffusa root methanolic extract could be used as a therapeutic drug against oral cancer.
Table 4: Patch dock score

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Figure 9: (a) Schematic 9, 12-Octadecadienoic acid [Z, Z]-. (b and c) Schematic reprsentations of BAD and Bcl-2 proteins in 3D structures, (b) 3D structure of BAD protein, (c) cartoon representation of Bcl-2 protein, (d and e) schmatic reprsentations of BAD and Bcl-2 proteins in 3D structures interact with compound-9, 12-Octadecadienoic acid [Z, Z]-respectively. (d) 3D structure of BAD protein with compound interaction (e) 3D structure of Bcl-2 protein with compound interaction

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 > Discussion Top


Plants being a rich source of phytoconstituents have attracted researchers to evaluate their phytochemical and pharmacological aspects. Boerhavia spp., a widely distributed plant found in the tropical and sub-tropical areas of Asia, Africa, America, and Australia are used in traditional medicine for the treatment of diabetes, stress, dyspepsia, abdominal pain, inflammation, jaundice, enlargement of spleen, heart diseases, bacterial infections, and impotence. The presence of assorted chemical compounds account for the significant biological activities seen in B. diffusa extracts. In this study, the presence or absence of major active phytoconstituents like alkaloids, anthraquinones, flavonoids, glycosides, reducing sugar, saponins, steroids, tannins, and terpenoids were compared between the aqueous and methanolic extracts of B. diffusa.

The present study suggested that B. diffusa root extracts had potential reducing power of the free radical. Flavonoids are one among the compounds present in abundance in the extract, which when ingested, are transformed to phenolic acids capable of annulling free radicals. Antioxidants are important in protecting cells from free radicals, and numerous studies have shown their cancer slowing or preventive effects.[12],[27] Our results showed that the BDME had higher reducing power compared with the BDAE. This finding was consistent with other studies where methanolic extracts of leaves of plants like Syzygium cumini proved to be more effective than other extracts.[28] Previously, BDME has been shown to have better radical scavenging capacity than n-hexane and ethyl acetate extracts.[29] Similarly, antioxidant properties of methanolic Cissus quadrangularis leave extract had higher reducing power activity compared with other extracts.[30] The free radical molecules can start chain reactions and can cause cell damage or cell death. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions. CAT and POD activity reflected the antioxidant potential of the extracts. CAT and POD are antioxidant enzymes localized in the peroxisome where it catalyzes the conversion of reactive hydrogen peroxide (H2O2) to water and molecular oxygen; its expression is tissue specific and highly variable. Excessive H2O2 can damage cells and their surrounding tissues; therefore, proper clearance of it is important. CAT and POD is downregulated in various tumors.[27],[31],[32]

PPs possess anticancer properties through mechanisms that include antioxidant, antiproliferative, and anti-inflammatory activities.[33] In addition, phytochemicals modulate intracellular signaling pathways that might have a role in apoptosis and cell-cycle arrest. Therefore, we looked at the cytotoxic effect on KB oral cancer cells. The present study suggested that BDME and BDAE exhibited potent cytotoxic effect against KB cells. Various extracts from Thymus caramanicus Jalas and Kalanchoe laciniata have been shown to have cytotoxicity against KB oral cancer cells.[34] The antioxidant and cytotoxic activities observed in this study can be attributed to the presence of assorted phenolic compounds. Phenolic compounds at a higher concentration could become a pro-oxidant and alter the redox balance of tumor cells. This surge in intracellular ROS promotes apoptosis through activation of caspase-3. Varying degrees of apoptosis have been achieved at different concentrations by many compounds. Early apoptosis of HeLa cells was achieved with essential oil from Atlantia monophylla.[35] Similarly, a derivative [3-Npc] from dihydro-pyranochromenes family showed both early and late apoptosis of K562 human leukemia cells.[36] However, the inability to use some of these compounds as a therapeutic on humans is due to their toxic nature. In this regard, BDME and BDAE can be expected to be safe for human use. In this study, early and late apoptosis of oral cancer KB cells was observed when cells were incubated with the extracts. The methanolic root extract of B. diffusa showed higher percentage of G0/G1 phase in increasing concentrations, whereas S phase and G2/M phase percentage decreased which confirms the lowering of DNA content. Aqueous root extract of B. diffusa showed lower percentage of G0/G1 phase, S phase, and G2/M phase in increasing concentrations. In addition, B. diffusa root extracts showed up-regulation of Bad (pro-apopototic protein) and downregulation of Bcl-2 (anti-apopototic protein) against oral cancer indicative of KB oral cell line apoptosis. It is well accepted that cytological investigations elucidate the anti-proliferative effect routed through membrane blebbing, membrane instability, and distressing the cytoskeleton of the cells by the extracts.

The active constituents of the extract penetrate into the mitochondrial outer membrane by permeabilization leading to the release of pro-apoptotic proteins and second mitochondria-derived activator of caspases/direct inhibitor of apoptosis [IAP]-binding protein, which ultimately leads to apoptotic cell death.[7],[37] A modification in the structure can be brought in the compounds which makes the compounds more potent. Computational studies indicated that a compound [9,12 octadecadienoic acid [Z, Z]-] present in the B. diffusa root methanolic extract was a suitable therapeutic option against oral cancer. If required, an additional modification in the structure could make the compound more potent. In summary, B. diffusa root extracts have been proved to possess good scavenging capacity of free radicals; higher concentrations of the extract exhibited cytotoxic effect on KB oral cancer cell line that induce apoptosis by regulating the anti-apoptotic proteins and by stalling the synthesis of DNA. Hence, we conclude that B. diffusa root methanolic extracts possess anticancerous activity against oral cancer KB cells, which in future, after validation of the obtained results by in vivo studies, may be used as a therapeutic drug.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

 
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