|Year : 2018 | Volume
| Issue : 4 | Page : 143-152
Anticancer Activity of Pancha Paasana Chendhuram on MCF-7 Cells
Venkatraman Manjari, Sannasi Murugesan, Vellaian Banumathi, Rajagopal Pattarayan
Department of Nanjunoolum Maruthuva Neethi Noolum, National Institute of Siddha, Tambaram Sanatorium, Chennai, Tamil Nadu, India
|Date of Web Publication||26-Dec-2018|
Department of Nanjumaruthuvam, National Institute of Siddha, Tambaram Sanatorium, Chennai 600047
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Siddha system of medicine is one of the ancient Indian medical systems that is popularly known for the management and treatment of chronic noncommunicable diseases. Herbs and minerals constitute the major part of Siddha medicines. Herbs, herbal extracts, and minerals are commonly used to treat various malignancies. Pancha Paasana Chendhuram (PPC) is a herbo-mineral formulation used to treat different types of cancers in Siddha practice. Methods: In the present study, we investigated the in vitro antioxidant and anticancer activities of PPC against breast cancer cells (MCF-7). Results: PPC showed moderate antioxidant effects; it also showed a dose-dependent inhibition of MCF-7 cell proliferation, with an IC50 (half maximal inhibitory concentration) of 59.26 mg/mL and nuclear damage in cancer cells. PPC upregulated Bax and Bcl2 and downregulated p53 mRNA (messenger Ribonucleic acid) expression in MCF-7 cells. Conclusion: These results suggest that PPC may be an interesting candidate for further studies in treating breast cancer.
Keywords: Anticancer, antioxidant, MCF-7, Pancha Paasana Chendhuram
|How to cite this article:|
Manjari V, Murugesan S, Banumathi V, Pattarayan R. Anticancer Activity of Pancha Paasana Chendhuram on MCF-7 Cells. Int J Nutr Pharmacol Neurol Dis 2018;8:143-52
|How to cite this URL:|
Manjari V, Murugesan S, Banumathi V, Pattarayan R. Anticancer Activity of Pancha Paasana Chendhuram on MCF-7 Cells. Int J Nutr Pharmacol Neurol Dis [serial online] 2018 [cited 2019 May 25];8:143-52. Available from: http://www.ijnpnd.com/text.asp?2018/8/4/143/248542
| Introduction|| |
Breast carcinoma is the most common malignancy diagnosed in women in recent times and the incidence rate is escalating alarmingly., It is one of the frequent causes of death in underdeveloped countries (324,000 deaths/year) and the second leading cause of cancer death in developed countries (198,000 deaths/year) after lung cancer. Thus, research in this field is important to overcome both economical and psychological burden. Female breast cancer patients have shown higher resistance and increased vulnerability to chemotherapeutic toxicity. Chemotherapeutic agents exert severe side effects, such as diarrhea, nausea/vomiting, leukopenia, anemia, alopecia, bone marrow depression, and hyperuricemia, teratogenicity, carcinogenicity, and fertility issues in men and women, and impaired cognitive functioning, such as difficulties with attention and memory, and executive functioning, such as fatigue and neuropathy. The major mechanism underlying these chemotherapy-induced lethal effects is believed to be caused by neuroinflammation and oxidative stress.,, Induction of apoptosis is the main criterion in cancer treatment as it destroys cells without causing inflammation. Many studies established that apoptosis is induced by various stresses, such as oxidative stress, endoplasmic reticulum (ER) stress, or chromatin damage. Oxidative stress created by the excessive generation of reactive oxygen species plays a critical role in the induction of apoptosis.
Due to quick disease relapse and severe side effects, most of the surgical resection, radiation therapy, and conventional chemotherapy are among the limited treatment options for breast cancer. Hence, there is a growing need and incessant urge to find new chemo-preventive agents with minimum or no side effects that may be effective in preventing and/or managing breast cancer and that also improve the quality of life in patients. Alternative therapeutic methods which are less toxic, eco-friendly, and inexpensive are widely being investigated. Herbal medicine is one of the best approaches to prevent, manage, and treat cancer that focuses on the naturally occurring chemical substances for improving health conditions, with fewer side effects in comparison with chemotherapy. Some of the herbs are well-known for their efficient antiproliferation and antimetastatic properties. The more notable drugs such as vincristine, vinblastine, and paclitaxel are Food and Drug Administration (FDA)-approved breast cancer drugs of plant origin. Many studies reveal that the possible mechanism underlying the anticancer effects of herbal medicine is mainly by boosting the immune system, inducing cell differentiation, inhibiting the activities of telomerase enzyme, and inducing apoptosis of cancer cells. Specifically, phytomedicine fight against the cancer cells by strengthening the immunity which in turn prevents the spread of cancer cells via inhibiting angiogenesis that feed the cancer cells, detoxifying the body via removing the free radicals, and cleanse the body from the toxic products.
Siddha system of medicine is one of the oldest Indian medicinal systems that possess evidences for both prevention and treating various types of cancers. In the Siddha literature, Chendhuram (red oxide form) has been reported to play key role in treating chronic ailments. It is prepared by making metallic salts or arsenical compounds into reddish fine powders by the process of burning, frying, calcinating, or grinding with various herbal juices or ceyaneers. Pancha Paasana Chendhuram (PPC) is one of the popular anticancer formulations in Siddha system of medicine. PPC contains herbal juices of Acalypha indica (kuppaimeni), Piper betle (vetrilai), Gossypium (paruthi), Enicostema axillare (vellarugu), Ocimum sanctum (thulasi), Pergularia daemia (veliparuthi), and Lippia nodiflora (poduthalai) mixed with minerals such as red sulfide of mercury (lingam), mercury subchloride (pooram), white arsenic (vellai pasanam), arsenic trisulfide (thalagam), arsenic disulfide (manosilai), sulfur (gandagam), and magnetic oxide of iron (gaantham). Till date, there is no scientific-based study to demonstrate the anticancer activity of PPC. The present study was designed to investigate the potential of PPC as anticancer agent against MCF-7 breast cancer cells, and its effects on the genes contributing mainly to the apoptotic cascades such as Bcl-2 (anti-apoptotic member), Bax (proapoptotic member), and p53 (tumor suppressor gene).
Many studies suggest that plant extracts act as prooxidants,,, and this effect can be exploited to kill cancer cells.
| Materials and Methods|| |
Chemicals and cell media
Dulbecco’s modified Eagles medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), 0.25% trypsin EDTA, antibiotics (penicillin, streptomycin), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ethidium bromide (EtBr), acridine orange (AO), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) stain were obtained from Hi-media Lab Ltd., Mumbai, India. Protease inhibitor cocktail, bovine serum albumin, acrylamide, 2-mercaptoethanol, sodium dodecyl sulfate, and N,N,N0,N0-tetramethylene diamine were purchased from Sigma-Aldrich (USA).
Procurement, identification, and authentication of raw drugs for Pancha Paasana Chendhuram
Red sulfide of mercury, mercury subchloride, white arsenic, arsenic trisulfide, arsenic disulfide, sulfur, and magnetic oxide of iron were procured from M/s. K. Ramasamy Chetty, Tranditional Medicine Shop, Chennai. All the mineral raw drugs were identified and authenticated by Department of Chemistry, Siddha Central Research Institute, Arignar Anna Govt. Hospital Campus, Arumbakkam, Chennai, India.
A. indica, P. betle, G. hirsutum, E. axillare, O. sanctum, P. daemia, and L. nodiflora were collected from forest area of Bairnaykanpatty—Dharmapuri Dt and Mathuranthagam—Kanchipuram Dt. All the herbals were identified and authenticated by Prof. Dr. P. Jeyaraman, PhD, Plant Anatomy Research Center, Sakthi Nagar, West Tambaram, Chennai, India.
Preparation of Pancha Paasana Chendhuram
Purified raw drugs were grinded with the above-mentioned seven herbal juices (which were added one after the other and cured for 12 h), until it attained a semisolid consistency. Then, the semisolid paste was made into small balls (villai) and dried under shade. Villai were placed on the betel leaves in a vessel made of mud. Mud vessel was surmounted with an equal-sized vessel rims sealed with clay plaster and winded for seven times. After drying, the whole set up was heated under a gentle flame (Deepam pol) for 12 h (on a stove) and then allowed to cool. The resulting product was grinded with A. indica juice for 12 h and again the processes of drying, heating, and cooling were continued. This process was repeated for another two times to get the final product “Chendhuram” form, which was collected, grinded well, and made into powered form. The final product is stored in a closed air-tight container.
In vitro antioxidant studies
DPPH radical scavenging assay of PPC
DPPH radical was assayed as described earlier. The reaction tubes contained 0.135 mM DPPH in methanol followed by 1.0 mL of PPC (0–1200 μg/mL). The mixture was kept in dark for 30 min at 37°C and the absorbance was read at 517 nm (Systronics India Pvt Ltd (2203), Ahmadabad, Gujarat, India). Quercetin was used as standard. The percentage inhibition was calculated by the following formula: DPPH (%) inhibition = [(ABS control−ABS sample)/(ABS control)] × 100.
Hydroxyl radical scavenging capacity of PPC
To 0.2 mL of different concentrations (160–3200 μg/ml) of PPC and catechin as standard, 0.2 mL of each solution (3 mM deoxyribose, 0.1 mM ferric chloride, 0.1 mM EDTA, 0.1 mM ascorbic acid, and 2 mM H2O2) were added and incubated at 37°C for 30 min. Then 0.2 mL of ice cold Trichloroacetic acid (TCA) (15% w/v) and Thiobarbituric acid (TBA) (1% w/v) was added. The reaction mixture was kept in a boiling water bath for 30 min and cooled, and the absorbance was measured at 532 nm.
H2O2 radical scavenging activity of PPC
Various concentrations (160–3200 μg/mL) of PPC and aliquot of 50 mM H2O2 were mixed (1:1 v/v) and incubated for 30 min at room temperature. After incubation, 10 μL methanol and 0.9 mL FOX reagent (prepared in advance by mixing nine volumes of 4.4 mM butylated hydroxytoluene (BHT) in methanol with one volume of 1 mM xylenol orange and 2.56 mM ammonium ferrous sulfate in 0.25 M H2SO4) was added to 90 μL of the incubated mixture. The reaction mixture was vortexed and incubated at room temperature for 30 min. The absorbance of the ferric-xylenol orange complex was measured at 560 nm. All tests were conducted in triplicate and gallic acid was used as standard. Percentage of inhibition was calculated using the formula: [(A0−A1)/A0] × 100, where A0 was the absorbance of the control (blank) F and A1 was the absorbance of compound.
Nitric oxide radical scavenging activity of PPC
Sodium nitroprusside (5 mM, 1.5 mL) in PBS was mixed in different concentrations of (200–6400 μg/mL) PPC and incubated at 25°C for 30 min. To the reaction mixture, 1.5 mL of Greiss reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride in 2% H3PO4) was added. The absorbance of the chromophore formed during diazotization of nitrite with sulfanilimide and subsequent coupling napthyl ethylene diamine was measured at 546 nm. Percentage radical scavenging activity of the sample was calculated as follows: percentage of inhibition−[(A0−A1)/A0] × 100, where A0 was the absorbance of the control (blank) and A1 was the absorbance in the presence of the compound.
In vitro anticancer activity
Human breast cancer MCF-7 cells were procured from National Centre for Cell Science, Pune, India. Growth inhibition of MCF-7 cells by PPC was determined by MTT assay. Cytotoxicity assay was used to determine the IC50 concentration of drugs or chemicals. MCF-7 cells were maintained as a monolayer culture in DMEM, supplemented with 10% FBS, in a humidified atmosphere at 37°C and 5% CO2. Cells were seeded at a density of 8000/well in 96-well microplate and treated with PPC (10–320 µg/mL) for 20 h. After drug exposure, 5 μL of MTT (5 mg/mL) dye was added to wells and wrapped with aluminum foil and incubated for 4 h. After incubation, medium was removed cautiously and 200 μL of DMSO was added to each well to solubilize the formazan crystals; it was then placed still in the dark for 15 min. MTT reduction was quantified by measuring the absorbance at 570 and 630 nm in enzyme-linked immunosorbent assay (ELISA) Plate reader (Bio-Rad Laboratories, Inc., Berkeley, California, United States). Each experiment was repeated at least three times.
Determination of apoptotic morphological changes
AO and EtBr staining were used to detect apoptotic cells affirmation. The cells were cultured in six well plate (3 × 104/well) treated with different concentrations of (10, 30, and 100 μg/mL) PPC for 24 h. The cells were fixed in methanol:glacial acetic acid (3:1) for 30 min at 4°C. Then cells were washed in PBS, and stained with AO/EtBr (1:1) for 30 min at 37°C. Stained cells were washed with PBS and viewed under a floid cell imaging station (Invitrogen, Waltham, Massachusetts, USA). The number of cells showing features of apoptosis was counted as a function of the total number of cells present in the field.
Reverse transcriptase polymerase chain reaction (RT-PCR)
MCF-7 cells (30 and 100 μg/mL) were washed twice with PBS. Two milliliter of TRIzol (Thermo Fisher Scientific, Carlsbad, California, United States) was added and transferred to centrifuge tube and vortexed. 0.2 mL of chloroform per 1 mL of TRIzol was added, and the tubes were shaken vigorously for 15 s and allowed to stand at room temperature for 5 min. The mixture was centrifuged at 10,000 rpm for 15 min and the aqueous phase was transferred to new tubes; 0.5 mL of isopropanol per 1 mL of TRIzol was added and mixed gently by inverting the sample five times and centrifuged at 10,000 rpm for 10 min. Supernatant was discarded and the RNA pellet was washed using 1 mL of 70% ethanol. Final pellets were dried by incubating in a dry bath for 5 min at 55°C. The pellet was then resuspended in 25 μL of Diethyl pyrocarbonate (DEPC)-treated water. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was conducted as described earlier by Techno Prime system to determine the levels of Bax, Bcl2, P53, and B-actin mRNA expressions. Primers [Table 1] were supplied by Eurofins Genomics, India.
Data were expressed as mean ± standard error of the mean (SEM). Linear regression analysis was applied to estimate the IC50 value. Mean difference between the groups was analyzed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison was used as post hoc test. Graph pad Prism 5.0 (San Diego, USA) software was used for statistical analysis.
| Results|| |
DPPH radical scavenging activity
PPC inhibited DPPH radical formation with an IC50 value of 533.2 μg/mL. The reference standard, quercetin, showed an IC50 value of 3.965 μg/mL [Figure 1].
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity of PPC is shown in Figure 2. IC50 value of PPC and gallic acid was found to be 1670 and 6.746 μg/mL, respectively.
H2O2 scavenging activity
H2O2 scavenging activity was assayed by FOX reagent method. PPC produced a dose-dependent inhibition of H2O2 with an IC50 value of 2222 μg/mL. IC50 value of gallic acid was found to be 59.5 μg/mL [Figure 3].
|Figure 3: Effects of PPC and gallic acid on hydrogen peroxide scavenging|
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Nitric oxide scavenging activity
PPC quenched nitric oxide formation in a dose-dependent manner [Figure 4]. PPC quenched 50% of nitric oxide with an IC50 value of 6218 μg/mL and that of curcuminoids was found to be 13.53 μg/mL.
Effect of PPC on cell viability
MCF-7 cells were treated with various concentration of PPC (10–320 μg/mL) [Figure 5]. Exposure to PPC produced a dose-dependent inhibition of MCF-7 cell growth in the tested conditions. Further, there were also morphological changes in the cells treated at higher concentrations. In MTT assay, the IC50 value of PPC was found to be 59.26 μg/mL.
|Figure 5: Effect of PPC on cytotoxicity (A) and morphological changes in control (B) and PPC 10 (C) and 320 (D) μg/ml|
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Apoptotic morphological changes
To determine the effect of PPC on nuclear and cytosolic status, double staining technique (AO and EtBr) was employed. Untreated cells were brightly fluorescent with green nuclei and normal morphology were seen [Figure 6]A. PPC at 30 and 100 μg/mL showed orange luminescent apoptotic body formation, when compared to control [Figure 6]B–D.
|Figure 6: Effect of PPC on apoptosis, acridine orange, and ethidium bromide dual staining (A) control, (B) 10, (C) 30 and (D) 100 μg/ml|
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Effect of PPC on mRNA expression pattern of apoptotic markers
RT-PCR analyses were performed to understand the role of PPC on Bcl-2 (apoptotic inhibitor), Bax (apoptotic promoter), and p53 (tumor suppressor) gene expression in MCF-7 cells. The internal control, β-actin, was used to normalize the Bax, Bcl2, and P53 gene expression. PPC-treated cells showed upregulation of p53 (100 μg/mL) and Bax (10–100 mg/mL) when compared to the untreated cells. A significant downregulation in the gene expression of Bcl2 was observed with PPC when compared to the untreated cells [Figure 7] and [Figure 8].
|Figure 8: (A) β-actin, housekeeping gene normalization of PPC at various concentration. (B) Effect of PPC on Bax and BCL2 mRNA expression. L1: Ladder; L2: Control—BCl2; L3: PPC (30) BCl2; L4: PPC (100) BCl2; L5: PPC (100) Bax; L6: PPC (30) Bax, and L7: Control-Bax|
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| Discussion|| |
The present study reveals that PPC exerts cytotoxic activity against human breast cancer cell lines. An earlier study showed that anticancer activity of Gowri Chinthamani Chendhuram containing the mixture of mercury and sulfur also produces antioxidant activity via induction of metallothionein. Rasagandhi mezhugu, which is prepared by the combination of mercury (Rasam) and sulfur (Gandhi), was clinically proved for its anticancer effect in patients with oral cancer. The biomedical organic chemistry (elemental medicine) categorized mercury as a chemotherapeutic element and sulfur as a nonmetallic essential element.
In the present study, PPC induced cell death of MCF-7 cancer cells in a dose-dependent manner. As cytotoxic activity is considered a prognostic anticancer effect, we investigated the apoptotic effects of PPC using AO and EtBr staining technique. Cell exposed to PPC showed increased apoptotic bodies, which further reveals that PPC produces nuclear damage and induces cellular death. In the present study, we speculate that arsenate and mercuric oxide used in PPC might have targeted cancer cells. Arsenic, a metalloid, exist as inorganic oxides or organic compounds, and its various forms are used in the Indian and Chinese medical systems in the treatment of various ailments including cancer for more than 2000 years. Arsenic compounds are finding important place in the treatment of various cancers, namely, breast cancer, glioma, hepatocellular carcinoma, cervical cancer, etc. Interestingly, the recent studies on nanoparticles/nanoliposomes showed better efficacy., But still more information on the toxicity of arsenic compounds has to be studied.
Among the several factors that contribute to the development of cancer, one of the cardinal features is the loss of balance between cell growth and cell death, resulting in increased cell proliferation, and the loss of removal of the damaged/dead cells through apoptosis. Apoptosis is commonly defined as programmed cell death, controlled by various set of genes, and is an essential process during cellular development playing a key part in the pathogenesis of diseases including cancer. Certainly, many research studies suggested that the loss of apoptotic inhibition in favor of cell proliferation is accountable for breast cancer commencement and development. Generally, cancer cells do not undergo apoptosis easily, as they have defects in their ability to activate the signaling pathways of death. Thus, an important target in the anticancer therapy is to activate the apoptotic pathways in the cancer cells. Apoptosis is characterized by cleavage of chromosomal DNA into oligonucleosomal fragments, which can be described by cell shrinkage, blebbing of the membrane, chromatin condensation, and nuclear fragmentation., According to this notion, many phytomedicines, including plants, vegetables, herbs, plant extracts, spices, and herbo-mineral combination have been proved to be effective source of anticancer drugs, and many trials proved that their antitumor effects are exerted mainly by inducing apoptosis.
Induction of apoptosis induces various changes in the nucleus of the cells. Hence, analyzing the nuclear morphology of apoptotic cells stained with AO/EtBr (AO/EB) under fluorescent microscopy can help us to confirm the cytotoxic effects. AO, as a cationic dye, penetrates normal and early apoptotic cells with intact membranes and shows up as a green fluorescence, whereas EB enters the membranes of nonviable cells and binds to damaged DNA or RNA of the late apoptotic and dead cells, emitting orange–red fluorescence. Therefore, dual AO/EB staining is able to detect and distinguish the normal, early, and late apoptotic cells along with their nuclear morphology., In our study, PPC induced apoptosis that was evidently shown by the formation of nuclear changes and nuclear fragmentation using the dual staining method under the fluorescent microscopy.
Bcl-2 family proteins play an important regulatory role in apoptosis and are the main targets of anticancer therapy, and the ratio of Bax:Bcl-2 proteins increases during apoptosis. Bax (Bcl-2 associated × protein) is a proapoptotic member of the Bcl-2 gene family and enhances apoptosis by competing with Bcl-2. In healthy mammalian cells, Bax is commonly found in the cytosol, but during the apoptotic process, Bax undergoes a conformational change and gets inserted into the outer membrane of mitochondria. Bcl-2 is a human proto-oncogene, which code for Bcl-2 protein; it is an antiapoptotic protein belonging to the Bcl-2 gene family. It is located in the membranes of endoplasmic reticulum, nuclear envelope, and the outer membrane of the mitochondria. Bcl-2 protein suppresses apoptosis by preventing the caspase activation either by preventing the release of cytochrome C from the mitochondria and/or by binding to the proapoptotic members and neutralizing their effects., Decrease in expression of Bcl-2 leads to apoptosis. Tumor suppressor gene, p53, prevents the development of cancer. p53 promotes apoptosis by elevating the expression of Bax and suppressing the expression of Bcl-2. However, mutations in p53 gene lead to the development of cancer by disturbing the complex network of molecular pathways; it is the most commonly mutated gene associated with cancer. In the present study, PPC samples have shown cytotoxic effects in MCF-7 cell line and clearly the apoptotic property by AO–EB staining. mRNA expression study results using semiquantitative RT-PCR analyses indicate the upregulation of Bax and p53 and downregulation of Bcl-2. Thus, these results suggest that PPC may induce the apoptosis of MCF-7 cells through the downstream effect of tumor suppressor gene.Free radicals are the main culprits in the initiation and aggravation of many diseases. For the assessment of antioxidant potential of compounds, multiple assays are deployed. Antioxidant assays vary in terms of principle and experimental conditions. The routine experimental methods study the effects of antioxidants in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. In DPPH assay, violet-colored DPPH solution is reduced to yellow-colored product, diphenylpicryl hydrazine, by the addition of PPC. Our results revealed that the high concentration of PPC is needed for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity when compared to quercetin. This effect may be due to the electron-transferring ability of the PPC. Direct interaction of the free radicals with hydroxyl radicals of DNA induce mutations, which results in DNA damage. Hydroxyl radicals are formed by incubating Fe3+–EDTA premixture with ascorbic acid and H2O2 at pH 7.4, causing 2-deoxy-d-ribose degradation and generating a malondialdehyde-like product. Addition of PPC to the reaction mixture removes hydroxyl radicals and prevents further damage. PPC showed hydroxyl radical scavenging activity in higher concentration dose when compared with quercetin. Hydrogen peroxide destructs cells over oxidation of lipids, protein, and DNA, and consequently cell death through apoptosis. Hydroxyl radical present in the hydrogen peroxide was reacted with biomolecules and cause the tissue damage and cell death. The scavenging activity of hydrogen peroxide by PPC could reduce the tissue damage as well as oxidative-related disorders. Nitric oxide is synthesized in the vascular endothelial cells, certain neuronal cells, and phagocytes. Chronic exposure to nitric oxide radical can cause various carcinomas and inflammatory conditions. Cancer cells exposed to PPC showed reduced oxidative stress by scavenging the DPPH, hydroxyl, hydrogen peroxide, and nitric oxide radicals due to the presence of potent antioxidant principles.
| Conclusion|| |
This study revealed the cytotoxicty and apoptotic inducing capacity of PPC against MCF-7 breast cancer cells. PPC regulate the key genes involved in apoptosis and thereby exerts anticancer activity. However, PPC showed only moderate to very low antioxidant properties. Further, investigations using in vivo models are recommended to understand tolerability and mechanism of action of PPC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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