INTRODUCTION:

Currently, a growing number of people worldwide are afflicted with cancer, a malignant disease brought on by fast and unchecked cell growth. Being the second greatest cause of death worldwide, cancer is a burden for many developing nations¹.

Hepatocellular Carcinoma (HCC) is the most common primary liver malignancy. Hepatitis C is a term used to describe the uncontrolled growth and spread of malignant cells in the liver. It is most commonly associated with chronic liver illnesses such as cirrhosis brought on by an infection with Hepatitis B or C^2,3. Among widespread solid tumours in humans, hepatocellular carcinoma (HCC) is the fifth most prevalent tumour and the fourth main cause of cancer-related death⁴. Effective treatments with increased immunological potential are desperately needed to counteract the current morbidity and mortality associated with HCC without endangering the host, given the increasing incidence of the disease. Thus, the development of new therapies for HCC is crucial⁵. A number of liver illnesses are accompanied with oxidative stress, which plays a role in the onset and development of liver damage⁶.

Treatment options for liver cancer include surgery, thromboembolisation with chemotherapeutic agents, and liver transplantation. Side effects of radiation and chemotherapy negatively affect the psychological, sociological, and physiological aspects of human being ⁷. In fact, mortality due to side effects of chemotherapy or radiation is more than the cause itself. Hence, there is an urgent demand for safe and promising therapeutic agents which can reduce progression of cancer with minor or no side effects.

Entada phaseoloides (Linn.) Merr. (Family: Fabaceae), a South Chinese creeper, has notable medicinal benefits. considering their curative attributes, stems and seeds are both employed in traditional Chinese medicine⁸. All parts of this plant is used in indigenous medicine; the general population in tropical and sub-tropical districts use for the treatment of a wide range of diseases, including hemorrhoids, stomachache, toothache, gastritis, and lymphadenitis⁹. Its anti-inflammatory, analgesic, anti-arthritic, anti-diabetic, hypo-lipidemic, antiulcer, anti-complement, antioxidant, and antimicrobial, in-vitro anticancer activities were reported^10-17. In fact, molecules obtained from natural sources—that is, "natural products"—such as plants, marine life, and microorganisms, have played a significant and ongoing role in the identification of leads for the creation of conventional medications that are used to treat the majority of ailments¹⁸

Previous studies on Phaseoloideside E against hepatic carcinoma were poorly understood. Phaseoloidin, isolated by our group, is a homogentisic acid glucoside from the seed kernel of Entada phaseoloides, whose structure has been established as homogentisic acid 2-O-I-D-glucoside¹⁹. The present study involves investigation of methanolic extract of seeds of Entada phaseoloides (MEEP) and Phaseoloidin (PHA) against hepatic carcinoma by exploring its cytotoxic effect and its potential mechanism for apoptotic induction.

MATERIALS AND METHODS:

Materials:

Chemicals and media:

DMEM, PBS, trypsin EDTA, trypan blue, DCFDA, MTT [3-(4,5-dimethylthiazole-2-yl)-2, 5-tetrazoniumbromide] were procured from Sigma Aldrich (MO, USA). FBS and antibiotic- antimycotic (100X), MitosoxTM Red were procured from Invitrogen (Life technologies). DMSO (Sigma, Aldrich) was used to dissolve PHA which was then diluted in cell culture media before use. All the primary and secondary antibodies were purchased from Santa Cruz Technology (Danvers, MA) and all the chemicals were of analytical grade.

Collection and authentication:

In the months of April and May of 2016, Entada phaseoloides seeds were purchased from the Sonapur local market, Kamrup (Metro), Assam. A voucher specimen (AAU-NW-EVM-3) was deposited and preserved in the herbarium of the Department of Agronomy, Assam Agricultural University, Jorhat-785013, Assam. It had been identified by Dr. Iswar Chandra Barua, Principal Scientist, Assam Agricultural University, Jorhat, Assam.

Extract preparation and Isolation of Phaseoloidin:

Extract was prepared as reported in our previous study ²⁰. Briefly, after removing the kernel from the seeds, they were shade dried, powdered mechanically, weighed and stored in air tight container. Subsequently a beaker containing 250g of powdered material was soaked in 1000mL of methanol for 72 h, with a sterile glass rod being used to agitate the mixture every 18 h. Whatman filter paper No. 1 was used to collect the filtrate three times. A rotary evaporator (BUCHI, R-210, Labortechnik AG, Meierseggstrasse, Switzerland) was used to remove the solvent at decreased pressure, leaving behind a dark brown residue (MEEP). It was kept until usage in an airtight jar at 4°C. It was found that the dry powder recovery percentage was 10.11% w/w.

Furthermore, using silica gel (60–120 mesh, 150×15 cm), five grammes of MEEP were run using column chromatography and eluted sequentially with CHCl3:MeOH (8:2) and CHCl3:MeOH (3:2) to provide four fractions(F1–F4). After the F3 fraction was further purified using column chromatography and eluted with CHCl3/MeOH (12:1), Phaseoloidin was obtained as a white, amorphous powder (15mg), which was identified using its mass and NMR spectra.

Cell culture:

HeLa, MDA MB 231, DU 145, A 549, and HepG2 cells were procured from American type culture collection (ATCC), USA. HeLa, HepG2, and MDA MB 231 cells were maintained in DMEM media, DU 145 was maintained in MEM media, and A549 maintained in F12K media. All cells were maintained with 10% fetal bovine serum, 1% penicillin/streptomycin. Sub-culturing was done, when cells reached a confluency of 70%.

Cell viability assay:

Cell viability was determined by MTT colorimetric assay with slight modification^21,22,23. Briefly, Cells were seeded onto 96-well plate and allowed to attach overnight in a CO₂ incubator. Cells were treated with 0.411–100 (μg/mL) PHA and MEEP for 24h, followed by 10μL of MTT (5mg/mL) in 100μL medium and incubated at 37°C for 4h. Then the media with MTT was removed, purple formazan crystals so formed were dissolved in 200μL of dimethyl sulphoxide (DMSO) and read at 570nm in a multi detection plate reader (Spectramax M4, 158 Molecular devices, USA). Cytotoxicity was expressed as the half-maximal inhibitory concentration (IC₅₀ value) of both MEEP and PHA. Growth inhibition was determined based on the percentage of control of the untreated cell. All the treatments was performed triplicate.

Detection of intracellular reactive oxygen species (ROS) generation (DCF-DA):

The fluorescent probe 2,7 -dichlorodihydrofluorescein diacetate (DCF-DA) was utilized to measure the changes of intracellular ROS levels assay with slight modification^24. Briefly, HepG2 cells were seeded into 6-well plates separately at a density of 4 × 10⁵ cells/2mL per well, after incubation with test compounds (MEEP 20 and 30μg/mL and PHA 30 and 50μM) for 24h, following cell collection, three PBS washes were performed and incubated with 10µM of DCF-DA in serum-free medium for 20min. After washing with PBS to remove the unconjugated probe, the samples were dispersed in 500µL of PBS and analyzed by flow cytometer (Attune NxT focusing cytometer, Life technologies Invitrogen Singapore) using excitation at 488 nm blue LASER and 530nm emission filter. Approximately 10,000cells were evaluated per sample excluding debris and cell clumps.

Measurement of mitochondrial super oxide generation:

Mitosox^TM Red is a mitochondrial super oxide indicator gives a red fluorescence in presence of super oxides. HepG2 cells were seeded into 6-well plates separately at a density of 4 × 10⁵ cells/2 mL per well and allowed to attach to well overnight. Cells were treated with 20 and 30 μg/mL of MEEP and 30 and 50μM PHA for 24h. Subsequent incubation, PBS was used to wash the cells and incubated with 100nM Mitosox^TM Red super oxide indicator in serum-free medium for 20min. After washing with PBS, trypsinised cells were re-suspended in 300μL PBS and analyzed by flow cytometer^25.

Measurement of mitochondrial membrane potential (DΨM):

The dual fluorescent dye JC-1 is a specific probe for the detection of alterations in mitochondrial membrane potential in living cells. JC1 binds to healthy mitochondria and aggregate there to produce red fluorescence, but in case of impaired mitochondria JC-1 stays in monomeric form to produce green fluorescence. Mitochondrial membrane potential (ΔΨm) assay was performed with slight modification^26. Briefly, HepG2 cells were seeded on 12-well plates at a density of 1×10⁵ cells/ mL, incubated overnight to allow adhesion. The HepG2 Cells were treated with 20 and 30μg/mL of MEEP and 30 and 50μM PHA for a period of 24h. The cells were thoroughly washed with PBS, trypsinised, centrifuged. 2μM JC1 dye was added to cell suspension, incubated for 30min and analysed by flow cytometry (Attune NxT, Thermo Fisher Scientific).

Western blotting:

Using western blotting, molecular mechanistic investigations were carried out. After treatment of HepG2 cell line with PHA at two different concentrations 30 and 50μM for 24h, cells were lysed with RIPA buffer, protease cocktail mixture, phosphatase inhibitor with slow rotation, and then centrifuged at 10,000rpm for 10 minutes at 4°C to obtain the supernatant. The protein concentration of the supernatant was quantified by Bradford reagent (Himedia) with Bovine serum albumin (BSA) as the standard. Samples with 40μg of total protein were mixed with an equal volume of 2X Laemmli buffer, boiled for 10min at 95°C cooled, loaded and separated in 12% polyacrylamide gels containing sodium dodecyl sulfate (SDS), and transferred on to the nitrocellulose (NC) membrane. Following one hour at room temperature blocking with 3% bovine serum albumin in 1X TBST, the membranes were incubated with the following primary antibodies for an entire night at 4°C: BAX (monoclonal antibody, dilution 1:1000); Bcl-2 (monoclonal antibody, dilution 1:1000); Caspase 3 (monoclonal antibody, dilution 1:1000); Cleaved caspase 3 (monoclonal antibody, dilution 1:1000); PARP (monoclonal antibody, dilution 1:1000); β-actin (monoclonal antibody, dilution 1:1000); (Santa Cruz Biotechnology, Inc. Anti-mouse and anti-rabbit peroxidase-conjugated secondary IgG antibodies were used to detect immunoreactivity for one hour at a dilution of 1:5000²⁷. Blots were detected using Chemiluminescence. The band intensities were quantified using Image J software (NIH, Bethesda, MD, USA).

Statistical Analysis:

The data gathered was displayed as the (mean±SEM) of three replicates. Using the Graph Pad Prism program, version 6.0, the variation between the different groups was assessed using one-way analysis of variance (ANOVA) and Dunnett's Multiple Comparison Test.

RESULT:

Extracts preparation:

The recovery percentage with respect to dry powder was found to be 10.11% w/w.

Identification of phaseoloidin from MEEP:

Phaseoloidin has been extracted and identified from MEEP, and the structure was verified using spectral data. 1H-NMR (DMSO-d6, 500 MHz) δ: 9.00(1H, s, 4-OH), 6.94 (1H, d, J = 8.6 Hz, H-3), 6.58 (2H, m, H-5, H-6), 4.99 (2H, brs, H-7), 4.53 (1H, d, J =7.4 Hz), 3.68 (dd, J = 11.8, 1.9 Hz, 1H), 3.61 (1H,d, J =15.7) and 3.51(1H,d,J=15.7),3.46 (dd, J = 11.8, 5.6 Hz, 1H),3.20 (td, J = 5.7, 3.2 Hz, 4H), 3.14 (1H,dd, J = 7.4 and 8.5). 13C-NMR(DMSO-d6, 300 MHz)δ: 172.92 (C-8), 152.02 (C-4), 148.51 (C-1), 125.88 (C-2), 117.45 (C-6), 117.03 (C-3), 113.97 (C-5), 102.91 (C-1'), 76.90 (C-5’), 76.49 (C-2’), 73.38 (C-3’), 69.74 (C-4’), 60.80 (C-6’), 34.93 (C-7). 329.0875 ([M-H]-;C14H18O9calcd. 329.0873) is the HRMS (ESI+) m/z.

Cytotoxicity assay for MEEP and Phaseoloidin:

In-vitro anticancer activity of MEEP was screened in various cell lines by MTT assay protocol. Initially, various cancer cells were exposed to different concentrations of MEEP for 48hrs. As shown in fig. 1, MEEP inhibited cancer cell line growth with IC₅₀ values of 108.5±1.38, 91.45±0.66, 80.56±2.56, 71.48±3.05, and 33.69±1.80μg/mL in HeLa, MDA MB 231, DU 145, A549, and HepG2 cell lines, respectively. Anticancer activity of MEEP was cell line specific, where HeLa cells showed less sensitivity and HepG2 cells showed higher selectivity (Fig. 1A). Further, Anti-cancer activity of isolated compound PHA was tested in HepG2 cell line where MEEP showed higher selectivity. As expected, 48 h of treatment with Phaseoloidin with HepG2 cell line showed dose dependant inhibition of cell viability with an IC₅₀ value of 47.4±3.3 μM (Fig.1B).

Fig. 1: A. Graphical representation of IC₅₀ values of MEEP in various cell lines B. Dose response curve of phaseoloidin on HepG2 cell line. Data represented as Mean ±SEM of triplicate independent experiments.

MEEP and Phaseoloidin induced Oxidative stress in HepG2 cell line:

In view of exploring the mechanism behind the anticancer activity, we have initially evaluated the effect of MEEP and PHA on oxidative stress in cancer cells. HepG2 cells were treated with 20 and 30μg/mL concentration of MEEP and 30 and 50μM concentrations of PHA for 48 h. DCFDA staining followed by flow cytometry confirmed that both extract and isolated compound showed dose dependant induction of ROS generation in HepG2 cell line.

Phaseoloidin upregulated intrinsic apoptotic pathway in HepG2 cell line:

As shown in figure 3, PHA treatment (30 and 50μM) triggered mitochondrial impairment followed by upregulation of Bax and down regulation of Bcl-2 (figure 4). Western blot analysis revealed that Ratio of Bax/Bcl-2 was increased in PHA treatment groups. Further, we have analysed the expression pattern of caspase and cleaved caspase, treatment with PHA induced the cleavage of caspase-3 dose dependently. Apoptosis was confirmed by the expression of cleaved PARP compared to PARP in treatment groups. PHA treatment activated the cleavage of PARP by intrinsic apoptotic pathway and subsequent activation of apoptosis (Fig. 4).

Fig. 4: Western blot analysis of PHA treated cells compared to control cells. Graph represents the ratio of BAX to Bcl-2, cleaved Caspase-3 to caspase-3, Cleaved PARP to PARP. All the values are Mean ± SEM (n=3), *p<0.05, **p<0.01, ***p<0.001, PHA treated groups compared with control group (One way ANOVA followed by Dunnett’s Multiple Comparison Test).

DISCUSSION:

Natural products are a well spring of anticancer medications which much of the time appear to be progressively compelling and additionally less harmful ²⁸. Approximately 60% of the chemotherapeutic drugs are the natural products from the plant origin^29. The crude plant extracts of Entada phaseoloides are reported to be useful against many cancer. It was also reported that through the production of reactive oxygen species, Phaseoloideside E, a novel naturally occurring triterpenoid saponin found in Entada phaseoloides, causes apoptosis in Ec-109 esophageal cancer cells^30. However, methanolic fraction and its isolated component phaseoloidin are not being studied for its anti-cancer property earlier. This prompted us to study in-vitro cytotoxicity of MEEP and PHA against few cancer cell lines. Cell death results from the up-regulation of the apoptotic pathway when ROS are produced in excess in cancer cells. Interestingly, MEEP (30μg/mL) and PHA (50μM) caused 10 fold reactive oxygen species generation in the HepG2 cell line as compared to the untreated cell line (fig. 2).³¹In previous reports we have elucidated the antioxidant potential of MEEP which is helpful for preventing inflammatory disorders³². This new role of MEEP and phaseoloidin as selective ROS inducers in cancer cell line can be used as a promising strategy to treat cancer without causing any side effects. Mitochondrial health is an important parameter for cell survival for any kind of cell. One thoroughly researched and promising avenue for cancer treatment is targeting mitochondria. Initiation of apoptotic signals in the intrinsic pathway depends on the mitochondrial membrane integrity^33. Most of the drugs causing induction of ROS are capable of impairing mitochondrial membrane potential, so here in this study we have evaluated the role of MEEP and PHA in induction mitochondrial stress and mitochondrial membrane potential by Mitosox^TM Red super oxide indicator and JC-1 staining. Cumulatively, MEEP and PHA augmented the generation of ROS and mitochondrial super oxides which can impair electron transport chain which further worsens the condition by releasing free radicals. Mitochondrial stress not only affects the energy production but also impairs the membrane potential. Impaired mitochondria can trigger apoptotic pathway by altering the ratio of apoptotic (Bax) and anti-apoptotic (Bcl-2 or Bcl-XL) proteins. Immunoblotting analysis revealed that PHA caused apoptosis in HepG2 cells through intrinsic apoptotic pathway. In cancer cells, apoptosis induction depends on ratio of Bax to Bcl-2³⁴. High expression of Bax is an indication of activated apoptotic machinery, whereas high Bcl-2 expression is an indication of cell survival. In proliferative stage, cancer cells tend to upregulate Bcl-2 expression in order to promote cell survival and drug resistance. The proteins of the Bcl-2 family are found on the outer membrane of the mitochondria, and their interactions regulate the permeabilization of the outer membrane, which releases cytochrome c into the cytoplasm and activates caspases. The caspase cascade is activated by the starting caspase (caspase 9), which cleaves caspase-3, which cleaves PARP and causes apoptosis.

CONCLUSION:

This is the primary investigation which showed in vitro anti-cancer activity of MEEP and Phaseoloidin against HepG2 cancer cell lines and proved molecular mechanism for induction of apoptosis in hepatic cell carcinoma cell lines. Moreover, we demonstrated that PHA induced HepG2 cell apoptosis in a caspase-dependent manner. Further investigations revealed that PHA induces cell death through the up-regulation of cellular ROS production. Future study may be directed towards in vivo study using xeno graft model.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors express sincere gratitude to Department of Biotechnology (DBT), New Delhi for sponsoring this work. This study is funded by Department of Biotechnology (DBT), New Delhi with project sanction no. BT/PR25221/NER/95/1082/2017 dated 13.8.2018. The authors express gratitude to Dr. K Suresh Babu (Centre for Natural Products and Traditional Knowledge) and Dr. Sistla Ramakrishna (Department of Applied Biology) at CSIR-IICT, Hyderabad, for their support with biological activities and compound characterisation. For the molecular study, the authors also acknowledge Dr. VGM Naidu of the Department of Pharmacology and Toxicology at the National Institute of Pharmaceutical Education and Research (NIPER) in Guwahati, Assam, India.

REFERENCES:

1. Karpagam T, Jannathul Firdous, Revathy, Shanmuga Priya, Varalakshmi B, Gomathi S, Geetha S, Noorzaid Muhamad. Anti-Cancer Activity of Aloe Vera Ethanolic Leaves Extract against In vitro Cancer Cells. Research J. Pharm. and Tech. 2019; 12(5): 2167-2170.

2. Dhandhukiya Manavkumar Kanubhai, Saiyed Musaratafrin Sabirali. Hepatocellular Carcinoma: A Review. Asian Journal of Research in Pharmaceutical Sciences. 2023; 13(2): 171-9.

3. Al-Shimaa M. Abas, Moustafa Salaheldein Abdelhamid, Noha Mohamed said, Marwa Sayed Sabea. Evaluation of clinical significance of kallistatin and macrophage inflammatory protein-1b for the diagnosis of liver cirrhosis and hepatocellular carcinoma in Egyptian patients. Research J. Pharm. and Tech 2019; 12(1): 43-49.

4. Mohamed Zerein Fathima, T.S. Shanmugarajan, S. Satheesh Kumar, B.V.Venkata Nagarjuna Yadav. Comparative in Silico Docking Studies of Hinokitiol with Sorafenib and Nilotinib against Proto-Oncogene Tyrosine-Protein Kinase(ABL1) and Mitogen-activated Protein Kinase (MAPK) to Target Hepatocellular Carcinoma. Research J. Pharm. and Tech. 2017; 10(1): 257-262.

5. Rahamat Unissa, Devaragattu Bhargav Varma, Naga Santhosh Kesana, Chekalta Srinivas, Baddanapally Shalem Raj. In Vitro cytotoxicity of L-Glutaminase against Hep-G2 cell lines. Research J. Pharm. and Tech. 2018; 11(6): 2213-2216.

6. Ashok Kumar K, Vijayalakshmi K, Saravanan D. Effect of Punica granatum peel and Vitis vinifera seeds on Biochemical Indices in DEN induced Hepatocellular Carcinoma in Rats. Research J. Pharm. and Tech. 2019; 12(6): 2758-2764.

7. Yang Q, Salim L, Yan C, Gong Z. Rapid Analysis of Effects of Environmental Toxicants on Tumorigenesis and Inflammation Using a Transgenic Zebrafish Model for Liver Cancer. J Mar Biotechnol. 2019; 21: 396–405. doi:10.1007/s10126-019-09889-8, PMID: 30852708.

8. Chandana Choudhury Barua, Sm Abdul Aziz Barbhuiya, Ajay Kakati, Lipika Buragohain, Syamprasad Np, Basweswar Gawali, Vgm Naidu. Methanolic extract of Entada phaseoloides inhibits Colorectal carcinoma cells proliferation of HT-29 cell by Modulating mitochondria-mediated apoptotic pathway. Research Journal of Pharmacy and Technology. 2022; 15(10):4439-6.

9. Mohan VR, Janardhanan K. Chemical and nutritional evaluation of raw seeds of the tribal pulses Parkia roxburghii g. Don. and Entada phaseoloides (L.) merr. Int J Food Sci Nutr. 1993; 44(1): 47–53. https://doi.org/10.3109/09637489309017422.

10. Dawane JS, Pandit VA, Rajopadhye BD. Experimental evaluation of anti-inflammatory effect of topical application of Entada phaseoloides seeds as paste and ointment. N Am J Med Sci. 2011; 3(11): 513-7. doi: 10.4297/najms.2011.3513, PMID: 22361498.

11. Dawane JS, Pandit VA, Rajopadhye BD. Anti-nociceptive effect of entada phaseoloides seeds formulation after topical application in arthritic wistar rats. J Clin Diagn Res. 2020; 7(12):2744–2746.

12. Kumari RR, More AS, Gupta G,Lingaraju MC, Balaganur V, Kumar P, et al. Effect of alcoholic extract of Entada pursaetha DC on monosodium iodoacetate-induced osteoarthritis pain in rats. Indian J Med Res. 2015; 141(4): 454.

13. Zheng T, Shu G, Yang Z, Mo S, Zhao Y, Mei Z. Antidiabetic effect of total saponins from Entada phaseoloides (L.) Merr. in type 2 diabetic rats. J Ethnopharmacol. 2012; 139(3): 814–821. https://doi.org/10.1016/j.jep.2011.12.025, PMID: 22212505

14. Lim TK, Lim TK. Entada phaseolides. In: Edible Med Non-Medicinal Plants. Netherlands:Springer, Dordrecht; 2012, p. 627–633.

15. Aktar F, Kuddus MR, Faruque SO, Rumi F, Quadir MA, Rashid MA, et al. Antioxidant, Cytotoxic, Membrane Stabilizing and Antimicrobial Activities of Bark and Seed of Entada phaseoloides (L.) Merr.: A Medicinal Plant from Chittagong Hill Tracts. J Pharm Nutr Sci. 2011; 1: 171–176.

16. Li K, Xing S, Wang M, Peng Y, Dong Y, Li X, et al. Anticomplement and antimicrobial activities of flavonoids from Entada phaseoloides. Nat Prod Commun. 2012; 7(7): 867–871.

17. Chih Liu W, Kugelman M, Wilson RA, Rao K V. A crystalline saponin with anti-tumor activity from Entada phaseoloides. Phytochem. 1972; 11(1): 171–173. https://doi.org/10.1016/S0031-9422(00)89984-7.

18. Alaspure R.N., Nagdeve S.R. Isolation of Active Constituent of Acorus calamus Rhizomes Extract and Evaluation of its Anti-cancer Activity. Research J. Pharm. and Tech. 2011; 4(12): Dec. 1825-1832

19. Nzowa LK, Teponno RB, Tapondjou LA, Verotta L, Liao Z, Graham D, Zink MC, Barboni L. Two new tryptophan derivatives from the seed kernels of Entada rheedei: effects on cell viability and HIV infectivity. Fitoterapia. 2013; Jun 1; 87: 37-42. doi: 10.1016/j.fitote.2013.03.017,PMID: 23537889.

20. Barua CC, Buragohain L, Purkayastha A, Saikia B, Babu KS, Kumari GS, et al. Entada phaseoloides attenuates scopolamine induced memory impairment, neuro-inflammation and neuro-degeneration via bdnf/trkb/nfκb p65 pathway in radial arm maze. Int J Pharm Pharm Sci. 2018; 10(9): 29; http://dx.doi.org/10.22159/ijpps.2018v10i9.27487.

21. Yuan J, Mehta PP, Yin MJ, Sun S, Zou A, Chen J, et al.. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity. Mol Cancer Ther. 2011; 10(11): 2189–2199. doi: 10.1158/1535-7163.MCT-11-0185,PMID: 21750219

22. Le Dinh Chac, Bui Bao Thinh, Nguyen Thi Yen. Anti-cancer activity of dry extract of Anoectochilus setaceus Blume against BT474 breast cancer cell line and A549 lung cancer cell line. Research J. Pharm. and Tech. 2021; 14(2): 730-734.

23. Carolin P, Mariappan A, Meena Kumari R. In vitro Evaluation of Anticancer Activity of Karanthai Legium (Kl) – A Siddha Medicine for Cervical Carcinoma against Hela Cell Line By MTT Assay. Research Journal of Pharmacy and Technology. 2024; 17(2): 807-0.

24. Shrivastava S, Kulkarni P, Thummuri D, Jeengar MK, Naidu VGM, Alvala M,et al. Piperlongumine, an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells. Apoptosis. 2014; 19(7): 1148–1164. doi: 10.1007/s10495-014-0991-2,PMID: 24729100.

25. Chen B, Li H, Ou G, Ren L, Yang X, Zeng M, et al. Curcumin attenuates MSU crystal-induced inflammation by inhibiting the degradation of IκBα and blocking mitochondrial damage. Arthritis Res Ther. 2019; 21(1): 1–15. doi: 10.1186/s13075-019-1974-z,PMID: 31455356.

26. Sivandzade F, Bhalerao A, Cucullo L. Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe. Bio Protoc. 2019; 9(1): e3128. doi: 10.21769/BioProtoc.3128, PMID: 30687773

27. Pal HC, Sharma S, Elmets CA, Athar M, Afaq F. Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: Role of mitochondrial membrane potential disruption and consequent caspases activation. Exp Dermatol. 2013; 22(7): 470–475.doi: 10.1111/exd.12181, PMID: 23800058

28. Gu LY, Chen Z, Zhao J, Ruan XJ, Zhao SY, Gao H, et al. Antioxidant, anticancer and apoptotic effects of the Bupleurum chinense root extract in HO-8910 ovarian cancer cells. J BUON. 2015; 20(5): 1341–1349.PMID: 26537084

29. Hamburger M, Marston A, Hostettmann K. Search for new drugs of plant origin. Adv Drug Res 1991; 20: 167–215. https://doi.org/10.1016/B978-0-12-013320-8.50007-1

30. Mo S, Xiong H, Shu G, Yang X, Wang J, Zheng C, et al. Phaseoloideside E, a novel natural triterpenoid saponin identified from Entada phaseoloides, induces apoptosis in Ec-109 esophageal cancer cells through reactive oxygen species generation. J Pharmacol Sci. 2013; 122(3): 163–175.

31. Kumar Jeengar M, Kumar S, Shrivastava S, N P S, L. Katanaev V, Uppugunduri S, et al. Niclosamide exerts anti-tumor activity through generation of reactive oxygen species and by suppression of Wnt/ β-catenin signaling axis in HGC-27, MKN-74 human gastric cancer cells. Asia-Pacific J Oncol. 2020: 1–13. doi: 10.32948/ajo.2020.08.06.

32. Barua CC, Buragohain L, Purkayastha A, Saikia B, Babu KS, Kumari GS, Barua AG. Entada phaseoloides attenuates scopolamine induced memory impairment, neuro-inflammation and neuro-degeneration via bdnf/trkb/nfκb p65 pathway in radial arm maze. Int J Pharm Pharm Sci. 2018; 10(9): 29.

33. Szilágyi G, Simon L, Koska P, Telek G, Nagy Z. Visualization of mitochondrial membrane potential and reactive oxygen species via double staining. Neurosci Lett. 2006; 399(3): 206–209. doi: 10.1016/j.neulet.2006.01.071, PMID: 16530963.

34. Arbab IA, Abdul AB, Sukari MA, Abdullah R, Syam S, Kamalidehghan B, et al. Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: A bioassay-guided approach. J Ethnopharmacol. 2013; 145(1): 343–354. doi: 10.1016/j.jep.2012.11.020, PMID: 23178663

Received on 29.07.2024 Revised on 15.11.2024

Accepted on 14.01.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3295-3301.

DOI: 10.52711/0974-360X.2025.00476

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.