Phenolics from Rubus fairholmianus induces cytotoxicity and apoptosis in human breast adenocarcinoma cells

Accepted Manuscript Phenolics from Rubus fairholmianus induces cytotoxicity and apoptosis in human breast adenocarcinoma cells Blassan P. George, Heidi Abrahamse, Nanjundaswamy M. Hemmaragala PII:

S0009-2797(17)30392-7

DOI:

10.1016/j.cbi.2017.08.005

Reference:

CBI 8072

To appear in:

Chemico-Biological Interactions

Received Date: 24 April 2017 Revised Date:

3 August 2017

Accepted Date: 7 August 2017

Please cite this article as: B.P. George, H. Abrahamse, N.M. Hemmaragala, Phenolics from Rubus fairholmianus induces cytotoxicity and apoptosis in human breast adenocarcinoma cells, ChemicoBiological Interactions (2017), doi: 10.1016/j.cbi.2017.08.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Apoptosis inducing property of Rubus methanolic column subfraction LCMS analysis of RFM

Solid tumor growth

Survival days 18.28±0.96 19.56±1.05

Lifespan(%)

Standard-10 mg/kg

33.48±1.98**

83.15

RFM-100 mg/kg RFM-50 mg/kg RFM-25 mg/kg

31.74±1.45* 29.08±2.17* 28.62±2.68*

73.63 59.08 56.56

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Groups Tumour control 0.1% CMC

MCF-7 cells

LDH

ATP

Caspase 3/7 activity

Cytochrome C release

ACCEPTED MANUSCRIPT Phenolics from Rubus fairholmianus induces cytotoxicity and apoptosis in human breast adenocarcinoma cells Blassan P. George*, Heidi Abrahamse and Nanjundaswamy M. Hemmaragala Laser Research Centre, Faculty of Health Sciences, University of Johannesburg,

Address:

Blassan P. George

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Corresponding Author:

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Doornfontein, 2028, Johannesburg, South Africa

Laser Research Centre,

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Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa

Telephone:

+27 11 559 6550

Fax:

+27 11 559 6558

E-mail address:

[email protected]; [email protected]

E-mail address:

Heidi Abrahamse: [email protected] Nanjundaswamy M. Hemmaragala: [email protected]

ACCEPTED MANUSCRIPT Abbreviations: Rubus fairholmianus root methanolic column subfraction, RFM; (2, 2-diphenyl-1picrylhydrazyl), DPPH; (2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)), ABTS; Trolox Equivalent Antioxidant Capacity, TEAC; Total Antioxidant Activity, TAA; Lactate Dehydrogenase, LDH; Adenosine 5’ Triphosphate, ATP; Reactive Oxygen Species, ROS;

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Organisation for Economic Co-operation and Development, OECD; Institutional Animal Ethics Committee, IAEC; Committee for the Purpose of Control and Supervision of Experiments on Animals, CPCSEA; Dalton's Lymphoma Ascites, DLA; Ehrlich Ascites carcinoma, EAC; Carboxy Methyl Cellulose, CMC; Increase in Life Span, ILS; Hank’s

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Balanced Salt Solution, HBSS; Propidium Iodide, PI. Abstract:

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Herbal medicine is an important part of health care system in most of the countries. Rubus fairholmianus is an unexplored berry in folkloric medicine. In this study, we aimed to understand the importance of R. fairholmianus in pharmaceutical industry for the development of cost-effective cancer therapeutic drugs using in vivo and in vitro analysis. Chemical characterisation, antioxidant, antiproliferative and apoptosis inducing properties of

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R. fairholmianus root methanolic column subfraction (RFM) were investigated. The RFM displayed the presence of alpha-tocopherol, flavonol glycoside and apigenin in the chemical characterisation. DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS (2, 2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assays exhibited an activity of 7.56

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µg/mL (IC50) and 20514.7 µM trolox equivalents/g respectively. The solid and ascites tumors in mice were reduced significantly upon 100 mg/kg RFM treatment by reducing the tumor volume (1.86 cm3), tumor weight (69%) and increasing life span (31.74 days). The

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morphological features of RFM treated MCF-7 cells showed the cell damage and decreased cell numbers. The viability of treated cells decreased with 67.73% at 20 µg/mL against 96.50% in untreated cells. The treated cells (20 µg/mL) resulted in a substantial decrease (p<0.001) in cellular ATP proliferation, increased the LDH cytotoxicity, increased apoptotic cells population (33.92%) and reduced the population of viable cells (Annexin V-/PI-) (45.56%). Increased caspase 3/7 activity and cytochrome c release were also observed in treated cells. This is the first evidence about in vitro and in vivo anticancer activity of R. fairholmianus phenolics. The major phenolics such as alpha-tocopherol, flavonol glycoside, and apigenin might be the reason behind the caspase-mediated apoptosis. Further work is warranted to study the individual effects of these bioactive compounds in the induction of cell

ACCEPTED MANUSCRIPT death. Due to the apoptosis inducing properties, it can be considered as an effective adjuvant therapeutic agent in clinical trials. Keywords: Rubus; apoptosis; antioxidant; MCF-7; breast cancer; cytochrome c

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1. Introduction

Cancer is a global burden with an estimated number of new cases every year is expected to increase from 10 million in 2002 to 15 million by 2025, with 60% of those cases

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occurring in developing countries. Natural products are important sources of anticancer agents. More than 60% of drugs for cancer treatment are of natural origin, particularly derived from plants. Several studies focused on the activity of natural compounds mainly

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phytochemicals in the prevention of many deceases including cancer; generally, they include vitamins and phenolic compounds. Modern research found that the phenolic compounds and the chromatographic fractions of many crude plant extracts have antitumor effects on many cancer cells [1-5]. Many such plant-derived agents have been developed and clinically used for the treatment of a variety of cancers, and a number of agents are currently in preclinical development [6]. Over the last 30 years, approximately 45% of all anticancer drugs have been

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derived directly or indirectly from plant compounds, of which 12% are natural products and 32% are semisynthetic derivatives of such natural products [7]. Malignancy of breast is a common cancer among females and the foremost cause of

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mortality among women globally. Several predisposing factors such as; genetic, hormonal and environmental are involved in the breast cancer progression. Even though the recent developments in cancer research have reduced the mortality rate of breast cancer patients, the

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available treatments are still inadequate. In addition to conservative cancer treatments, chemoprevention using natural products is an effective preventive approach, which aims to decrease the incidence and mortality rates [8]. One of the mechanisms by which chemopreventive agents act is through their antioxidant property. These agents protect the cells from the toxic effects of reactive oxygen species (ROS), generated endogenously or exogenously, through their free radical scavenging actions and thus enhance the cellular capacity to combat oxidative stress [9]. This project was concentrated on antioxidant, antiproliferative, and apoptosis inducing potentials of the methanolic column chromatographic subfraction of Rubus fairholmianus root, an ethnomedicinally important tropical plant from Western Ghats of

ACCEPTED MANUSCRIPT India. Earlier, we reported the in vitro and in vivo bioactivities of root of this plant using various analgesic, anti-inflammatory and wound healing models. The correlation between antioxidant properties and pharmacological effects of Rubus extracts with special attention to inflammatory related deceases including cancer has been studied based on the folkloric knowledge. R. fairholmianus root extract also showed in vivo and in vitro antitumor effects

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on MCF-7, A375, A549 and Caco-2 human cancer cell lines [10-14]. In spite of many studies on its various biological properties, little information is available about the column fractions of this species to cytotoxic and apoptosis induction in MCF-7 cells. Therefore, this study designed to examine the DPPH and ABTS radical scavenging properties, cell viability and

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cytotoxicity on MCF7 breast cancer cells. In addition, we studied the flow cytometric analysis to find out the apoptotic populations along with caspase 3/7 and cytochrome c release activities. The results would be important because there is very limited information

2.

Materials and methods

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available on the activity of the column sub fractions of Rubus.

2.1. Plant material, extraction and fractionation

R. fairholmianus (common name: Molucco raspberry; synonym: R. moluccanus) was collected from Mannavan Shola forest of Marayoor, Kerala, India and the plant material was

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authenticated by Botanical Survey of India (BSI), Southern Circle. A voucher specimen was deposited in the herbarium of BSI for future comparisons (No. BSI/SRC/5/23/20102011/Tech.1657). The shade dried, powdered root of R. fairholmianus Gard. was extracted in Soxhlet apparatus using non-polar to polar solvents following the hot percolation method.

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Based on the preliminary antioxidant and phytochemical screening, the dried acetone extract was selected for further isolation of phytochemicals. During the chromatographic isolation of

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bioactive compounds, the methanolic column subfraction (RFM) showed highest antioxidant properties. This column fraction also possessed oncoprotein and inflammatory protein inhibitory activities and yielded two phenolic compounds (3-(Iminomethyl)-2, 4dimethylphenol and 3-Methylbutyl benzoate) [15]. 2.2. Fourier Transform Infrared Spectroscopy (FTIR) and Liquid Chromatography– Mass Spectrometry (LCMS) characterization of RFM FTIR spectroscopy measurements were recorded by making pellets of the powdered extracts with potassium bromide (1:10) and the pellets were passed through Infrared beam and the readings were obtained after adjusting blank background. The mass spectroscopy of

ACCEPTED MANUSCRIPT the extracts was recorded using Shimadzu LCMS-2020 after dissolution with methanol (1:10) by electrospray ionization method. 2.3. In vitro antioxidant activities 2.3.1. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and Trolox equivalent

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antioxidant capacity (TEAC) Blois [16] and Re et al. [17] methods were followed for DPPH and 2, 2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays respectively. DPPH scavenging activity was expressed in IC50. In ABTS assay, the unit of total antioxidant

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activity (TAA) is the concentration of trolox having equivalent antioxidant activity expressed as µM/ g sample extracts.

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2.4. Cell culture

Human breast cancer- MCF-7 (ATCC HTB-22) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) medium complemented with Fetal Bovine Serum (FBS) (10%) (FBS; Gibco 306.00301), penicillin/streptomycin (1%) (PAA Laboratories GmbH, P11-010) and 1 µg/mL Amphotericin B (PAA Laboratories GmbH, P11-001). Cells were cultured at 37ºC with 5% CO2, 80% humidity and subcultured weekly twice. When the cells

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become confluent, were washed with Hank’s Balanced Salt Solution (HBSS, Invitrogen, 10543F) and dissociated using 1 mL/25 cm2 of TryplExpress (Gibco, 12604). Cells then, seeded (5 x 105 cells/plate) in 3.5 cm2 diameter culture plate and incubated for 6 h to attach.

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2.5. In vivo solid and ascites tumor studies

The animal studies have been performed as per the standard OECD (Organisation for

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Economic Co-operation and Development) guidelines and the study received approval from the Institutional Animal Ethics Committee (IAEC) for the Committee for the Purpose of Control

and

Supervision

of

Experiments

on

Animals

(CPCSEA)

(Reg.

No.

KMCRET/Ph.D/03/2011). Dalton's Lymphoma Ascites (DLA) cells and Ehrlich Ascites Carcinoma (EAC) cells were cultured and maintained in vivo in Swiss albino mice by the intraperitoneal transplantation of cells in phosphate buffer saline. The tumor cells were aspirated by phosphate buffer saline from the peritoneal cavity of mice and were counted using trypan blue viability method. Finally, the desired concentration of the cells were administrated subcutaneously or intraperitoneally to mice for the solid and ascites tumor growth.

ACCEPTED MANUSCRIPT DLA cells (2 x 105 cells/animal) were injected subcutaneously to the hind limb of Swiss albino mice for solid tumor growth. The animals were grouped as; Group I- treated with standard cyclophosphamide (10 mg/kg b. wt.), Group II- IV- RFM (25, 50 and 100 mg/kg b. wt.) doses, Group V-untreated and VI- 0.1% CMC (Carboxy Methyl Cellulose). After 24 h of tumor inoculation, the diameter of the hind limb was noted and treatments were

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continued for 10 days. Tumor volume was measured every fifth day for up to 40 days. The tumor volume, V = 4/ 3π d12 d2, where d1 and d2 are the major and minor diameters. The percentage reduction of tumor weight was calculated, % Inhibition = 1-C/T x 100, C is average tumor weight of untreated animals and T for the treated groups.

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Male Swiss albino mice were injected with EAC cells (2 x 105 cells /animal) intraperitoneally for ascites tumor growth. The animals were treated for 10 days with RFM

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(25, 50 and 100 mg/kg b. wt. concentrations) and cyclophosphamide (10 mg /kg b. wt.) after 24 h of tumor inoculation. The ascites tumor growth was noted and increase in life span (ILS) of the animals were calculated, % ILS= (T-C)/C x 100, T and C is the average number of days the treated and control animals survived [18]. 2.6. Cell morphology-inverted microscope

The cellular morphological changes of cells after the treatments were carefully noticed

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using Wirsam, Olympus CKX 41 inverted light microscope after 24 h of incubation with RFM (5, 10 and 20 µg/mL). 2.7.

Cell viability, cytotoxicity and metabolism Trypan blue assay (Sigma-Aldrich T8154) used for checking the viability percentage

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of cells. Equal volume (10 µL) of cell suspension and 0.4% trypan blue were mixed and loaded to a hemocytometer and counted using automated cell counter (Countess™

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Automated Cell Counter, Invitrogen). The Cyto-Tox96 X assay (Anatech, Promega G 400) used to measure cytotoxicity. The membrane integrity was assessed by quantifying the LDH released to the culture media. Equal volume (50 µL) of LDH reagent and cell culture medium mixed and incubated in dark at room temperature for 30 min and colorimetric compound was read at 490 nm (Perkin-Elmer, VICTOR3™). The CellTiter-Glo1 luminescent assay (Promega, G7571, Anatech Analytical Technology, South Africa) followed for the quantification of ATP as a direct measure of cell metabolism. Equal volume (50 µL) of ATP reagent and cell suspension was mixed and incubated at room temperature for 10 min in dark and the luminescence was read using 1420 Multilabel Counter Victor3 (Perkin-Elmer, Separation Scientific).

ACCEPTED MANUSCRIPT 2.8. Flow cytometry analysis The Annexin V- fluorescein isothiocyanate (FITC) apoptosis detection kit (Becton Dickinson, 556570, Scientific Group, South Africa) used to distinguish the population of apoptotic and non-apoptotic cells. The cell suspension (1 x 106/mL) (100 µL) was stained with equal volume (5 µL) of Annexin V and propidium iodide (PI), vortexed and incubated

(FACS) Aria flow cytometer (BD bioscience). 2.9. Caspase 3/7 activities

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for 10 min at room temperature in dark and run in Fluorescence Activated Cell Sorting

Caspases 3/7 activities was analysed using the Caspase-Glo 3/7 luminescent assay kit

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(Promega G8091, Whitehead Scientific, Bracken fell, South Africa). Equal volume (50 µL) of treated cells and Caspase reagent was seeded in 96-well luminous plate (Scientific Group

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Adcock Ingram, Midrand, South Africa BD354651), incubated for 3 h at room temperature and the luminescent signal was read using the Victor3 (Perkin-Elmer, Separation Scientific). 2.10.

Cytochrome c release assay

An apoptogenic component, cytochrome c is required for apoptotic events. An Enzyme linked immunoassay (ELISA) (human cytochrome c Platinum ELISA kit) was used

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to detect the level of cytochrome c in the cytosol. The re-suspended cells were spun for 5 min at 2200 rpm and then lysed. The lysate was centrifuged for 15 min at 1200 rpm and a 50-fold dilution of the supernatant was done with 1 x assay buffer. Lysate were further diluted (1:2); a volume of 100 µL sample was added to each wells and blank wells contained 1x assay

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buffer. Fifty microliters of biotin-conjugated anti-human cytochrome c antibody was added and incubated for 2 h at room temperature. Thereafter, the plate was washed three times with

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400 µl wash buffer and Streptavidin-HRP secondary antibody (100 µL) was added and incubated. After the wells were washed, 100 µL of tetramethyl-benzidine (TMB) substrate was added and incubated at room temperature for 10 min. Finally, the reaction was stopped with stop solution (100 µL) and absorbance was measured at 450 nm using the Victor3 microplate reader (Perkin-Elmer). 2.11.

Data analysis and statistics

SigmaPlot version 13.0 was used for statistical analysis. All treatments were compared with control groups by one-way ANOVA to analyse the statistical significance. Significance between control and experimental groups are expressed as p<0.05 (*), p<0.01 (**) and p<0.001 (***).

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3. Results 3.1. Chemical characterization of RFM: FTIR, LC MS

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It is well known that, majority of plant derived organic compounds are bioactive; especially, when the active functional group is a derivative of glycosidic linkage, the overall drug would be very potent. In case of methanolic subfraction of R. fairholmianus root, the same trend was observed while characterizing by Infrared spectroscopy (Figure 1) and Liquid chromatographic-mass spectrometry (Figure 2). The predominant Infrared peaks observed at

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3270, 1606, 1048, and 794 cm-1. The peak at 3270 indicates the C-H stretching of aromatic ring, 1606 corresponds either to ether linkage or carbon-carbon double bonds, 1048

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corresponds to carbon-carbon stretching, and 794 for C-H bending frequencies. These observations giving an idea of RFM having organic compounds with aromatic ring, phenolic group, ether linkage, C-C single bonds and C-C double bands. Furthermore, the LC-MS peak interpretations of components in the Rubus extract indicated the molar masses to be 469.71, 779 and 893.

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Figure 1: FTIR analysis of Rubus fairholmianus methanolic column subfraction of root

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Figure 2: LC-MS analysis of Rubus fairholmianus methanolic column subfraction of

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root

3.2.Antioxidant-DPPH and ABTS

ACCEPTED MANUSCRIPT The stable free radicals such as DPPH and ABTS were effectually scavenged by RFM. DPPH radical scavenging activity was expressed as IC50 values compared to the standard antioxidants Rutin and Quercetin and ABTS by trolox equivalents (Table 1). RFM scavenged the DPPH radicals (IC50: 7.56 ± 1.43) compared to the standards (IC50: 5.98 ± 1.43 and 4.62 ± 1.55 for Rutin and Quercetin, respectively). Likewise, RFM considerably

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scavenges the ABTS radical at 20514.7 ± 59.1 µM trolox equivalents/ g. Table 1: Antioxidant, viability, cytotoxicity and proliferation assays. Groups

ABTS- TEAC assay (µM trolox equivalents/ g)

20514.7 ± 59.1

79.58 ± 0.97 **

10 µg/mL 20 µg/mL 5 µg/mL

74.13 ± 0.99 **

10 µg/mL 20 µg/mL 5 µg/mL

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10 µg/mL

Quercetin 4.62 ± 1.55

Control

96.50 ± 0.65

67.73 ± 1.02 ** 0.5067 ± 1.61 ** 0.5156 ± 1.25 ** 0.5302 ± 2.32 ** 399145.54 ± 5826.21 *

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LDH cytotoxicitymembrane integrity (A490nm)

5 µg/mL

Rutin 5.98 ± 1.43

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Trypan blue viability (%)

ATP proliferation(LuminescenceRelative Light Unit)

RFM 7.56 ± 1.43

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Assays DPPH (IC50 µg/mL)

0.4250 ± 4.13

574932.67 ± 5361.46

341281.11 ± 5038.43 **

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20 301923.29 ± µg/mL 5283.21 ** Values are expressed as mean ± SE (n=6) for viability, cytotoxicity and proliferation assays; mean ± SD (n=6) for antioxidant assays. The significant differences between treated and controls groups are shown as *p<0.05, **p<0.01. (RFM- R. fairholmianus root methanolic column subfraction; DPPH- 2,2-diphenyl-1-picrylhydrazyl, ABTS- 2,2′-azinobis-(3ethylbenzothiazoline-6-sulfonic acid), TEAC- Trolox Equivalent Antioxidant Capacity, LDH- Lactate Dehydrogenase; ATP-Adenosine 5’ Triphosphate) 3.3. In vivo antitumor activities Solid tumor volume of mice treated with RFM was found considerably reduced compared with control animals. Tumor volume of control animals and CMC treated animals on 40th day was 3.95 and 3.53 cm3 whereas tumor volume of animals treated with 100, 50 and

ACCEPTED MANUSCRIPT 25 mg/kg b. wt. RFM was 1.86, 2.22 and 2.82 cm3 respectively on 40th day (Figure 3). The multiple scatter plot (Figure 4) shows the difference in percentages of tumor weight after the treatment. There is a significant decrease observed in RFM treated groups compared to vehicle control group (9.5%). The standard drug, cyclophosphamide found to be highly effective in reducing the weight of solid tumor with the highest inhibition percentage (81.5%,

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P<0.001). However, RFM treated groups also reduced the tumor weight significantly (69.00, 54.00 and 49.75% for 100, 50 and 25 mg/kg b. wt. respectively). The effect of RFM on ascites tumor growth is shown in Table 2. All animals in control and vehicle control groups died of tumor by 18.28 and 19.56 days. Cyclophosphamide (10 mg/kg) was found to be the

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most active and increased the life span of animals by 33.48 (p<0.01) while RFM 100, 50 and 25 mg/kg showed 31.74, 29.08 and 28.62 (p<0.05) days of increase in lifespan. Figure 3: Effect of RFM on solid tumor growth

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Tumor volume

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1 0

n=6

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10th

15th 20th 25th 30th 35th Cyclophosphamamide (10 mg/kg.b.wt.) RFM (100 mg/kg. b. wt.) Control RFM (50 mg/kg b. wt.) RFM (25 mg/kg b. wt.) 0.1% CMC (Vehicle control)

40th

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Table 2: Effect RFM on ascites tumor growth.

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Groups Mean survival days Increase in lifespan (%) Tumor control 18.28 ± 0.96 0.1 % CMC treated animals 19.56 ± 1.05 Cyclophosphamide (10 mg/kg b. wt.) 33.48 ± 1.98** 83.15 RFM (100 mg/kg b. wt.) 31.74 ± 1.45* 73.63 RFM (50 mg/kg b. wt.) 29.08 ± 2.17* 59.08 RFM (25 mg/kg b. wt.) 28.62 ± 2.68* 56.56 Values are expressed as mean ± SE (n=6) and significantly different at * p<0.05 and ** p<0.01 compared to tumor control. RFM- R. fairholmianus root methanolic column subfraction. CMC: Carboxy Methyl Cellulose (vehicle control) 3.4. Microscopy

The morphological variations of RFM treated cells were compared with the control cells (Figure 5). The treated cells observed irregular with loss of intact membrane, even if the lower concentration (5 µg/mL) viewed intact. While at 10 and 20 µg/mL concentrations the RFM treated cells showed significant changes in morphology. The significant variations such as loss of intact membrane, cell detachment from the plate and change of morphological features were evident when compared to untreated cells. The important morphological

ACCEPTED MANUSCRIPT features of apoptosis were observed by inverted light microscopy in RFM treated MCF-7 cells.

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Figure 5: Morphological changes of MCF-7 cells after RFM treatment

Viability, cytotoxicity and metabolism The cell viability was assessed by trypan blue viability test, this method helped to

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calculate the viable and dead cells percentage (Table 1). There was a decreased number of viable cells after RFM treatment. The control cells showed 96.50% viability; whereas RFM

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treated cells showed 79.58, 74.13 and 67.73% for 5, 10 and 20 µg/mL, respectively. The decrease in viability percentage was significant with all tested concentrations (p<0.01). The membrane damage of MCF-7 cells after the treatment with RFM was measured by the release of LDH by following CytoTox961 Assay. The control cells showed lesser LDH release. The RFM treatments triggered the release of LDH than the control cells. A significant (p<0.01) dose dependant rise in LDH release was observed at increasing concentrations of RFM (Table 1). The energy level in MCF-7 cells remained higher, which was noticeable from the increased ATP level in control cells. Exposure of MCF-7 cells to the RFM caused a reduction in the intracellular ATP pool, which indirectly depicts the decreased cellular metabolism. The cells incubated with increasing concentrations (5, 10 and 20 µg/mL) of RFM resulted a

ACCEPTED MANUSCRIPT significant decrease (p<0.05 and p<0.01) (Table 1) in cell metabolism compared to control MCF-7 cells, this is an indirect measure of cell proliferation. 3.5.1. Analysis of apoptosis: Annexin V-FITC staining, Caspase 3/7 activities and Cytochrome c release

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Annexin V/PI double staining distinguished the apoptotic and nonapoptotic cells population. This test was done to confirm whether the decrease in cell viability and proliferation, increase in cytotoxicity observed was due to apoptosis. The population of apoptotic cells increased with increase in concentration of RFM. The control cells remained

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in the live cell range quadrant during flow cytometric analysis. The RFM treated cells showed an increased apoptotic cell population and the non-apoptotic or necrotic cells concentration were found to be very low (Figure 6). Out of the four flow cytometric runs, one

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result close to average value has shown in Figure 6 as dot plot graph. The Annexin V+/PI+ (necrotic/ non-viable) population was less (1.04%) in control cells compared to RFM treated groups (5, 10 and 20 µg/mL of RFM showed 1.48, 13.61 and 19.87% respectively). The apoptotic (Annexin V+/PI-) cells population increased (5, 10 and 20 µg/mL of RFM showed 23.91, 30.20 and 33.92% respectively), whereas the viable cells (Annexin V-/PI-) population

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decreased (5, 10 and 20 µg/mL of RFM showed 74.64, 55.65 and 45.56% respectively) concurrently upon the RFM treatment. The upper right quadrant (Q2) represents the necrotic cells, which are positive for Annexin V binding and PI uptake. The lower right quadrant (Q4) represent the apoptotic cells, they are positive for Annexin V while PI negative,

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demonstrating cytoplasmic membrane integrity. The upper left quadrant also represents the

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dead cells, which are devoid of Annexin V, and take up the PI.

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The caspase 3/7 activity was assayed to further distinguish the mode of apoptosis induced by RFM on MCF-7 cells. The inhibition of cell growth in ATP proliferative assay, LDH toxicity, loss of viability and increase in early and late apoptotic cell population in

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response to RFM treatments was due to caspase dependent pathway. There was a significant increase in caspase activities (p<0.05 and p<0.01 for 10 and 20 µg/mL) in treated cells with increased concentrations of RFM (Figure 7) compared to the lower activity in untreated cells,

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probably due to the small amount of apoptotic cells in the growing cell population. The results showed that RFM increases the activation of caspase 3 and 7 indicating the potential

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mediator in apoptosis induced cell death. Figure 7: Caspase 3/7 activities

ACCEPTED MANUSCRIPT Cytochrome c release was determined after 24 h of RFM treatment by ELISA and the level was compared with control cells. Cytochrome c release from the mitochondria is a critical event during the execution of cell death. The ELISA results revealed that, the control cells were unable to elicit the damaging event to mitochondria and was not able to release cytosolic cytochrome c (Figure 8). Whereas the RFM treated groups (5, 10 and 20 µg/mL)

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were able to significantly (p<0.001) initiate cell damage and thereby release of cytochrome c.

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4. Discussion

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Figure 8: Cytochrome c release

Our previous studies indicate that the R. fairholmianus is potent in controlling the

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growth of breast, lung, colorectal cancer and melanoma cells in vitro and it has showed many in vivo pharmacological properties. Based on the strong in vitro and in vivo antitumor activities, root acetone extract was selected for the isolation of bioactive compounds responsible for the particular activities. R. fairholmianus root acetone extract was fractionated and run in silica gel column chromatography to isolate the bioactive compounds. Among these sub fractions isolated during chromatography, the methanol sub fractionated pooled fraction-16 showed highest antioxidant and inhibitory activity against inflammatory and oncoproteins and yielded two phenolic compounds [15]. This subfraction (R. fairholmianus methanolic column subfraction- RFM) was selected for testing the anticancer activities using in vivo and in vitro models. The recent researches are focused on the bioactive properties of column fractions since the active fractions contains many phytochemicals and the synergistic

ACCEPTED MANUSCRIPT action of these active principle compounds imparts many biological activities including antiproliferative and

antioxidative.

Recently many authors

[19-25]

reported

the

antiproliferative, anti-inflammatory and antioxidant properties of various fractions and isolated compounds of Rubus species. The extensive scanning of reports available in the literature on phytochemical studies

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hinted out the presence of flavonoids such as quercetin, kaempferol, caffeic acid and chlorogenic acid in Rubus. Additionally, phenolic acids, sugars, pectins, carboxylic acids, anthocyanins, catechins, vitamin C and saturated or unsaturated fatty acids were also reported [26-29]. As per the data obtained from LC-MS analysis of RFM, it is predicted that the molar

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mass 469.71 is attributed to alpha-tocopherol whose structure consists of benzene ring bearing hydroxy group and cyclic ether group attached with saturated carbon chain. The

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molar mass 779 indicated the presence of compound having 3-O-[6”’-p-coumaroyl glucosyl(1-2) rhamnoside derivative of quercetin (flavonol glycoside). In addition, peak at 893 indicated the presence of Apigenin 7-O-(2’’-dihydrogalloyl)-glucosyl-8-C-rhamnosyl-6-Cglucoside. Therefore, all these characterizations supported that RFM contain compounds such as alpha-tocopherol and glycosidic derivatives of flavonols. These compounds imparts the antioxidant properties to the subfraction. RFM showed significant antioxidant activities in

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DPPH and ABTS radical scavenging assays. There are other reports from closely related Rubus species, which also showed DPPH and ABTS radical scavenging activities. Nevertheless, our study revealed that RFM has good antioxidant activities measured by single electron transfer assays such as DPPH and ABTS as revealed by both methods and the

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comparison with the literature [30-33]. More studies are required to clarify the exact antioxidant mechanism(s).

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Recently many researchers focus on in vivo studies to confirm the activities of many

drugs including herbal formulations. The bioavailability and effect of drugs on the metabolic systems can easily be analysed using animal models. The in vivo results from this study indicated strong therapeutic potential of Rubus subfraction. Both tumor models used were very fast growing with ability to grow in most of the mice strains. The implantation of these tumors triggers local inflammatory reactions and resulting the formation of tumors since the ascites fluid is the vital source of nutrients for the tumors to grow quickly. There are some reports showing the induction of solid and ascites tumor in mice using DLA and EAC mouse cancer cells and checking the effects of various plant extracts, bioactive compounds, synthesised drugs and chemotherapeutic agents [34-37]. Our results showed the RFM

ACCEPTED MANUSCRIPT subfractions were active against DLA and EAC tumors when injected in to mice both subcutaneously and intraperitoneally. The tumor volume and weight decreased significantly in solid tumors and in ascites, the life span of the tumor bearing mice increased compared with the control animals. The viability of cell is associated with its metabolic functions and the assumption is

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that the damaged cell membranes are permeable. The negatively charged trypan blue dye cannot react with the intact cell membrane; therefore, the viable cells do not take up the blue stain [38]. From our results, it was clear that the viability of the MCF-7 cells decreased considerably upon the RFM treatment. To further understand, the cell viability and membrane

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damage, we have analysed the LDH leakage from the treated cells. The damaged cells are incapable of retaining the intracellular enzymes including LDH. The results shown, that the

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membrane integrity of the treated cells were considerably reduced which lead to the high LDH release and this analysis was complemented by the results of ATP cell metabolism assay and the morphological examination of the treated cells. The treatment with RFM induced considerable LDH leakage due to loss of membrane integrity. Which depicts the cytotoxic action of RFM on MCF-7 cells. The advantage of LDH assay is that which measures the cell death directly by physical disruption of cell membrane. ATP and LDH are

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the important biomarkers for cellular metabolism and cytotoxicity. These enzymes are high in metabolically active cells and upon damage; the cytosolic LDH will be released to the cell culture media. The actively dividing cells will show higher ATP levels than the damaging cells [39]. RFM treated cells showed a reduced ATP cellular metabolite activity or the

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cellular energetics depicting the decreased cellular metabolism and proliferation. RFM treatment possibly induced cytotoxicity by triggering apoptosis, and therefore changes in

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cellular morphology were used to demonstrate the occurrence of apoptosis. The results shown in Figure. 5 indicate that after treatment with RFM, cells change their morphology becoming rounded, and besides started detaching from the culture plate, losing its membrane integrity and shape of the cells. The chromatin condensation and nuclear fragmentation are to be confirmed by further analysis. These results are clear evidence that RFM induced apoptosis process [40]. Similar effects have been reported in the apoptosis induced by parthenolide in multiple myeloma and prostate cancer [41]. Apoptosis process maintains/regulates the homeostasis and dismissal of damaged cells. The deregulation of programmed cell death (PCD) contributes to cancer and other autoimmune diseases. The cells undergoing apoptosis is clearly distinct from others in

ACCEPTED MANUSCRIPT morphological features and the hallmark of apoptosis are the exposure of phosphatidylserine on the cell surface during the early phase, followed by membrane blebbing and nuclear fragmentation [42]. As induction of tumor cell apoptosis is the ultimate goal of cancer, our results showed that the apoptosis is triggered significantly by RFM treatments. Based on the previous studies, the polyphenolic compounds of R. fairholmianus impart its cytotoxic

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effects. Polyphenolic compounds have been reported to possess a range of biological properties including antitumor, antibacterial and antimutagenic. Apoptosis, the genetically controlled programmed cell death is an important marker for the evaluation of potent anticancer agents of natural origin. This is a consequence of a highly complex sequential flow

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of actions needed for the homeostasis. Hence, induction of apoptosis is considered a potential therapeutic approach to prevent cancer progression [43]. In our study, the results expressed that RFM induced apoptosis in MCF-7 cells. The Annexin V/PI double staining presented in

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our study gives a clear idea about the induction of apoptosis by RFM. One of the crucial features of cell death via apoptosis is the formation of protein complexes, which ultimately leads to the activation of initiator caspases, which further process the executioner caspases and lead to the cell death. Mitochondria, the powerhouse of cells plays a vital role on intrinsic/mitochondrial apoptosis pathway, the key component in mitochondrial apoptosis

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pathway is the inflow of cytochrome c from the mitochondria to the cytoplasm. The proapoptotic drugs which act on the mitochondrial membrane enhances the release of cytochrome c to the cytoplasm and where it forms complex with other apoptosis proteins and subsequently activates the caspase 3. Caspase 3 is considered the most important executioner

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caspase, which was activated by the initiator caspases. Initially the cytoplasmic cytochrome c complex activates the effector caspase 3 and followed by the activation of other members of

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caspase family leading to apoptosis [44-46]. The involvement of caspase-3 in RFM-induced apoptosis in human cancer cells is not

well known and this study strongly suggested that RFM induced apoptosis in MCF-7 cells. Our results show that the treatment with RFM increases the caspase 3/7 activity and leads to the release of cytochrome c, which indicates that RFM induces the caspase dependant apoptosis. The Annexin V/PI flow cytometric analysis was further supported by the caspase 3/7 activities and cytochrome c release from mitochondria. Many studies showed the property of plant extracts, column fractions, and the isolated bioactive compounds in inducing the apoptosis via caspase upregulation and by the release of cytosolic cytochrome c by drop in mitochondrial membrane potential [47-48; 44]. Various plant-derived compounds initiated

ACCEPTED MANUSCRIPT mitochondria-mediated pathway or the intrinsic apoptotic pathway, which is characterized by the loss of mitochondrial membrane potential and the release of cytochrome c from mitochondria. Bcl-2 family apoptotic proteins can initiate this pathway. Among the Bcl-2 family proteins, Bax and Bcl-2 have been identified as the major regulators of apoptosis [49]. Our results clearly demonstrated that the initiation of mitochondrial pathway of apoptosis by

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the release of cytochrome c from mitochondria, which indirectly correlated with the drop in mitochondrial membrane potential, thereby releasing the enzyme to cytoplasm. The consecutive activation of caspase family members is considered the prerequisite of apoptosis. Cytochrome c in cytosol participates in the activation of caspase-9, which in turn lead to the

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activation of executioner caspase-3 and cleavage of PARP, a specific substrate for caspase-3 [50]. In the present study, RFM treatment induced the activation of caspase-3 and caspase-7

mitochondrial apoptotic pathway. 5. Conclusion

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and these results suggested that RFM induced MCF-7 cell death is probably through intrinsic

In conclusion, this study confirmed that methanolic column subfraction of R. fairholmianus root is rich in phenolic compounds like alpha-tocopherol, flavonol glycoside and apigenin. RFM showed immense antioxidant properties by scavenging the DPPH and

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ABTS radicals. The RFM treatments effectively reduced the cell viability, ATP proliferation and increased the release of LDH from MCF-7 cells. The morphological examination was in support of LDH cytotoxic activity of RFM. The in vivo studies were also suggestive for the antiproliferative activities of RFM in solid and ascites tumor models. The cytotoxic analysis

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was linked to the apoptosis inducing abilities of RFM by showing more apoptotic cells population and decreased percentage of viable cells in Annexin V/PI double staining. This

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can be correlated with caspase 3/7 upregulation and cytochrome c release indicating the stimulation of caspase dependant apoptosis. This study provides supportive evidence that methanolic column subfraction of R. fairholmianus root has great potential to be a source for the development of novel healing agent against mammary tumor. Conflict of interest The authors confirm that this article has no conflict of interest. Acknowledgments

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Figure 1: Fourier Transform Infra-Red (FTIR) spectroscopy analysis of R. fairholmianus

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root methanolic column subfraction (RFM). The predominant Infrared peaks at 3270, 1606, 1048, and 794 cm-1 gives an idea of RFM having organic compounds with aromatic ring, phenolic group, ether linkage, C-C single bonds and C-C double bands. Figure 2: Liquid chromatographic-mass spectrometry (LC-MS) analysis of R. fairholmianus

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methanolic column subfraction of root. The predominant LC-MS peaks at 469.71, 779 and 893 indicated the mass equivalent to that of alpha-tocopherol and glycosidic derivatives of

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flavonols.

Figure 3: Effect of RFM on solid tumor growth shown in graph. A dose dependent decrease in the tumor volume was observed. The control and vehicle (0.1% CMC-Carboxy Methyl Cellulose) treated groups showed 3.95 and 3.53 cm3 tumor volume on 40th day. Which was quite high compared to the cyclophosphamide (10 mg/kg b. wt.) (1.05 cm3) and the RFM 100, 50 and 25 mg/kg b. wt. treated groups (1.86, 2.22 and 2.82 cm3 respectively). RFM: R. fairholmianus root methanolic column subfraction. Figure 4: Effect of RFM against DLA induced solid tumor weight. Significant (p < 0.001) percentage of reduction in tumor weight was observed in RFM treated groups (69, 54 and 49.75% for 100, 50 and 25 mg/kg b. wt. respectively). However, Cyclophosphamide found to

ACCEPTED MANUSCRIPT be highly effective in reducing the weight of solid tumor with the highest inhibition percentage (81.5%). RFM: R. fairholmianus root methanolic column subfraction. The significant differences between treated and controls groups are shown as ∗∗∗ p < 0.001. Figure 5: Morphological changes of MCF-7 cells after RFM (R. fairholmianus root methanolic column subfraction) treatment. There were no significant visible differences in

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control (a) and 5 µg/mL RFM treated groups (b), the cells did not show any cellular shrinkage and loss of cell number after the treatment. However, more dead cells observed at higher concentrations of RFM (10 and 20 µg/mL). The treated cells (c and d) showed loss of intact membrane, loss of contact with neighbouring cells, condensed and detached from the

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culture plates.

Figure 6: Annexin V FITC/PI staining used to assess the mode of cell death in MCF-7 cells. RFM (R. fairholmianus root methanolic column subfraction) treated MCF-7 cells showed an

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increased percentage of apoptotic population after 24 h incubation. The population of apoptotic cells in control group found to be lower (2.76%) compared with experimental groups; the attached table shows the statistical data of flow cytometric analysis. Figure 7: Effect of RFM (R. fairholmianus root methanolic column subfraction) on caspase 3/7 activities of MCF-7 cells. Caspase 3/7 activity determined as a function of caspase

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dependent apoptosis in cells after the 24 h treatment. There was a significant (p < 0.05 and p < 0.01) increase in caspase 3/7 activity in after 10 and 20 µg/mL RFM treatments compared to control. The significant differences between treated and controls groups are shown as ∗∗ p < 0.01 and ∗ p < 0.05.

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Figure 8: Effect of RFM (R. fairholmianus root methanolic column subfraction) on cytochrome c release. Cytochrome c release is an important measure of cellular damage.

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RFM treated groups showed significant (p < 0.001) release of cytochrome c compared with the control group. The significant differences between treated and controls groups are shown as ∗∗∗ p < 0.001.

ACCEPTED MANUSCRIPT Highlights This study confirmed that RFM is rich in phenolic compounds

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RFM effectively reduced the cell viability and ATP proliferation

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Morphological examination was supportive for the LDH cytotoxicity

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Decreased percentage of viable cells in Annexin V/PI double staining

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Caspase 3/7 and cytochrome C activities supported caspase dependant apoptosis

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