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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1235-1249

Phytochemical fractions from Annona muricata seeds and fruit pulp inhibited the growth of breast cancer cells through cell cycle arrest at G0/G1 phase


1 Centre of Excellence in Molecular Biology and Regenerative Medicine (DST-FIST Supported Centre), Department of Biochemistry (DST-FIST Supported Department), JSS Medical College, JSS Academy of Higher Education and Research (JSS AHER); Department of Molecular Biology, Yuvaraja's College, University of Mysore, Mysuru, Karnataka, India
2 Centre of Excellence in Molecular Biology and Regenerative Medicine (DST-FIST Supported Centre), Department of Biochemistry (DST-FIST Supported Department), JSS Medical College, JSS Academy of Higher Education and Research (JSS AHER), Mysuru, Karnataka, India
3 Centre of Excellence in Molecular Biology and Regenerative Medicine (DST-FIST Supported Centre), Department of Biochemistry (DST-FIST Supported Department); Special Interest Group in Cancer Biology and Cancer Stem Cells (SIG-CBCSC), JSS Medical College, JSS Academy of Higher Education and Research (JSS AHER), Mysuru, Karnataka, India

Date of Submission14-Jul-2019
Date of Decision12-Dec-2019
Date of Acceptance30-Dec-2019
Date of Web Publication18-Dec-2020

Correspondence Address:
Devananda Devegowda
Centre of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education and Research, Mysore, Karnataka
India
SubbaRao V Madhunapantula
Centre of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education & Research (JSS AHER), Mysuru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_494_19

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 > Abstract 


Introduction: Annona muricata (L.) (AM), commonly known as Soursop and Lakshmanaphala/Hanumaphala in India, has been extensively used in ethnomedicine for treating tuberculosis, urinary tract infections (UTIs) and cancers. The fruit is a rich source of antioxidants and antitumor agents.
Methods: In this study, we have extracted phytochemicals that exhibited anti-cancer property from the (a) fruit pulp using methanol (AMPM) and water (AMPW); and (b) seeds using methanol (AMSM). Qualitative phytochemical analysis showed the presence of phenolics, tannins, alkaloids, flavonoids, sterols, terpenoids, carbohydrates and proteins in AMPM and AMPW. All three extracts were first checked for in vitro antioxidant and anti-inflammatory properties and then tested for efficacy against MCF-7 and MDA-MB-231.
Results: Among these three extracts, AMSM showed the highest antioxidant power as well as ~80% inhibition at 320μg/ml concentration in both cell lines upon treatment for 24h. However, only about 40% inhibition was observed with 320μg/ml AMPM treatment, despite its highest anti-inflammatory potential. Water extract AMPW exhibited about 80% growth inhibition at 50% dilution. Since fruit pulp is the one consumed, the extracts AMPM and AMPW were further tested for apoptosis induction and cell cycle arrest. Analysis of the data showed increased apoptosis and G0/G1 cell cycle arrest upon exposure to AMPM and AMPW.

Keywords: Acetogenins, anticancer, anti-inflammatory, apoptosis, cell cycle arrest, Lakshmanaphala, Soursop


How to cite this article:
Prasad SK, Veeresh PM, Ramesh PS, Natraj SM, Madhunapantula SV, Devegowda D. Phytochemical fractions from Annona muricata seeds and fruit pulp inhibited the growth of breast cancer cells through cell cycle arrest at G0/G1 phase. J Can Res Ther 2020;16:1235-49

How to cite this URL:
Prasad SK, Veeresh PM, Ramesh PS, Natraj SM, Madhunapantula SV, Devegowda D. Phytochemical fractions from Annona muricata seeds and fruit pulp inhibited the growth of breast cancer cells through cell cycle arrest at G0/G1 phase. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1235-49. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1235/303895




 > Introduction Top


Accounting to 27% of all cancers in women, breast cancer is the most common cancer in India. The incidence and mortality rates due to breast cancer are rising in India and across the globe. The age-adjusted incidence rate and mortality rate of breast cancer, in India, were found to be 25.8 and 12.7 per 100,000, respectively. Furthermore, one out of every two women, newly diagnosed with breast cancer, dies of it in India.[1],[2],[3],[4],[5]

Use of complementary and alternative medicine is gaining importance in the modern era due to increased health benefits compared to other medical practices.[6] This growth has drawn the interest of the present-day scientific community, resulting in extensive studies on herbal medicines. Therefore, it is necessary to understand and identify the therapeutic as well as the toxic side effects of herbal remedies.[7],[8]

Annona muricata (Linn.), commonly known as Soursop or Lakshmanaphala/Hanumaphala in Kannada language of Karnataka, India, is a fruit-bearing tree, standing 5–6 m tall.[9] Leaves are evergreen and malodorous, with the upper surface being dark-green, glossy, and smooth, while the surface beneath is of a light-green shade. Flowers are plump, short-stalked, and triangular-conical in shape, with three fleshy, yellow-green outer petals and three pale-yellow inner petals.[10] The plant bears an oval- or heart-shaped fruit which is lopsided, curved, or sometimes irregular. Covered with a spiny, leather-like yet tender skin, the fruits are dark-green when raw and yellowish-green upon ripening. Pulp is granular and cream colored, enclosing a soft core. The pulp carries a unique musky, subacid to acid flavor.[11] Indigenous to the tropical regions of the Americas and Southeast Asia, A. muricata has been naturalized. It is now widely distributed across the tropical and subtropical regions of the world, including Karnataka in the Indian subcontinent [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d.
Figure 1: Photographs of (a) Annona muricata tree; (b) Annona muricata leaves; (c) Annona muricata fruit, and (d) Annona muricata flower

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Phytoconstituents

According to Gavamukulya et al., A. muricata has been found to contain 212 bioactive compounds that include acetogenins (AGEs), alkaloids, and phenolic acids.[9] AGEs are least investigated, polyether compounds found exclusively in the plants belonging to Annonaceae family. They comprise an aliphatic chain containing 32–34 carbons, attached to a terminal lactone or butenolide ring. Epoxide, hydroxyl, ketone, tetrahydrofuran (THF), and tetrahydropyran groups may also be featured in AGEs. In the A. muricata species alone, more than 120 AGEs have been reported. A THF ring is found in most of the AGEs constituting A. muricata.[12] Biomedical properties and toxicity of A. muricata are attributed to the presence of AGEs.[13],[14] Alkaloids, phytocompounds with basic nitrogen atoms, are found at highest concentration within the leaves. These nitrogenous organic compounds are found across the plant kingdom. They are known for their pronounced biomedical properties. Reticuline, coclaurine, atherospermine, stepharine, and coreximine, while anomurine, and anomuricine are classified as the major and minor alkaloids, respectively, found in A. muricata.[15] Meanwhile, phenolic acids such as gallic acid are also reported present in the leaves of A. muricata. Reports also indicate the distribution of as many as 37 such aromatic acid compounds across the plant. Nonetheless, lipophilic antioxidants, i.e., tocopherols and tocotrienols, were found exclusive to the fruit pulp.[16] Furthermore, carotenoids, amides such as N-p-coumaroyl tyramine, vitamins as well as cyclopeptides, are also reported to be present.[17],[18],[19] In addition to the above, the fruit pulp contains numerous volatile compounds, mostly esters of the both the aromatic and aliphatic types, as well as sesquiterpene-derived essential oils.[20],[21]

Biomedical properties

The social history and medicinal use of A. muricata have been highlighted by several studies.[9],[22],[23] A. muricata has been used extensively in various ethno-medicinal practices. Parts of the plant that include leaves, roots, barks, fruits, and seeds are being used to treat various diseases and disorders.[24],[25],[26] Since the 1940s, scientists have been studying the properties of A. muricata, validating its use in natural medicine.[27] Decoctions or juices of the different parts have been used in alleviating complications pertaining to liver, heart, kidney,[28],[29] fever,[30] spasms,[31] diarrhea, rheumatic disorders, neuralgia,[32] respiratory illness,[33] gastrointestinal problems,[26],[34] diabetes,[35],[36],[37],[38] jaundice,[24] and malaria.[39],[40] Furthermore, records indicate that A. muricata has been extensively used as a sedative,[41],[42] lactagogue, smooth muscle relaxant, nervine,[31] astringent,[43] parasiticide,[44] and an insecticide.[45] In the recent years, A. muricata has gained popularity as a treatment for hypotension,[25],[26],[46] hypoglycemia,[25] and range of cancers.[47] Across the timeline, numerous bioactive compounds have been identified.[9]

Annonaceous acetogenins, exclusive to the Annonaceae family of the plant kingdom, have been the principal bioactive compounds. Annonaceous acetogenins have been the key phytochemical agents responsible for the anticancer activity of the plant. As observed by Gavamukulya et al. and Tundis et al., the above class of phytochemicals was found to be more potent as compared to the prescribed chemotherapeutic drugs.[48],[49] The cytotoxicity observed in tumor cells is mainly attributed to the physiological activities of these AGEs, leading to the inhibition of mitochondrial complex and therefore resulting in apoptosis.[12],[50],[51] Nonetheless, ethanol extracts of the A. muricata leaves were reported to downregulate epidermal growth factor receptor, an oncogene overexpressed in breast cancer,[52] meanwhile Parama Astirin et al. reported upregulation of tumor suppressor protein p53 in cervical cancer cell lines, by the AGEs.[53] Dai et al. also reported the A. muricata fruit extracts for selective cytotoxicity, inhibiting the growth of MDA-MB-468 cells, having a greater Epidermal growth factor receptor (EGFR) overexpression, and not the normal breast epithelial MCF-10A cells.[52] Bullatacin, an AGE, was found to be 300 times more potent than paclitaxel, in reducing ovarian carcinoma in xenograft mice models, coupled with better selective toxicity as compared to the latter.[54] Ethyl acetate extracts of A. muricata pericarp were demonstrated by Jaramillo et al. for their potent tumoricidal properties against colorectal and lung cancer cell lines.[55] In a sole study conducted on testing antileukemic property of the A. muricata phytochemicals, A. muricata leaves ethanol extract was reported to upregulate the pro-apoptotic caspase-3, in K562 chronic myelogenous cell line for leukemia, and confirmed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay.[56] A. muricata leaf and stem extracts were found to effectively control nonmelanoma skin cancer (NMSCs), the leading cause of skin cancer-related complications and death. In a 2018 study conducted by Chamcheu et al., investigations revealed dose-dependent specificity against the UW-BCC1 and A431 NMSC cell lines, respectively, by cdk upregulation-induced cell cycle arrest at the G0/G1 phase and subsequent apoptosis induction, along with the suppression of smoothen homolog precursor (Smo), glioma-associated oncogenes (Gli 1 and 2), and sonic hedgehog (Shh), gene components of the activated hedgehog (Hh) pathway.[57] In addition, various studies ascertain potent tumoricidal properties of A. muricata phytochemicals against hepatoma, pancreatic adenocarcinoma, and prostate cancer.[58],[59],[60],[61] Medicinal uses of all the identified bioactive compounds have been extended, with a few toxicities reported. Nonetheless, not much is reported about the key phytochemicals responsible for inhibiting the growth of cancer cell lines. Hence, in this present study, an attempt was made to fractionate the A. muricata pulp and the kernel and to study the effect of fractionated phytoextracts on the growth inhibitory activity using in vitro -cultured cells.


 > Methods Top


Plant samples: Procurement and preparation of Annona muricata fruit and seed extracts

Samples of A. muricata fruits were procured from an organic farm at Kyathanahalli village in Mandya district, Karnataka, India (geographical coordinates: 12.46°N, 76.65°E). The fruits were identified and authenticated at the Agricultural Technology Information Center, Indian Council of Agricultural Research – Indian Institute of Horticulture, Bengaluru, India. Details of the authentication of the fruit sample can be found enclosed as an [Annexure 1]. The fruits were cut and separated into fruit pulp and seeds. The fruit pulp was freeze-dried for a week before being ground to a powder using a mixer grinder. The seeds were also ground into fine powder. Later, water and methanol extracts of the fruit pulp and methanol extract of the seeds were obtained as given below.



  1. Methanol extract of A. muricata fruit pulp: 60 g of freeze-dried fruit pulp in 300 ml methanol was subjected to Soxhlet extraction for 4 h at 50°C. The mixture was filtered using Whatman filter paper No. 1. The unfiltered material was air-dried, while the filtrate obtained was concentrated using a rotavapor and labeled A. muricata pulp methanol extract (AMPM)
  2. Water extract of A. muricata fruit pulp: 10 g of fruit pulp was added with 100 ml of distilled water and filtered. The filtrate was labeled A. muricata pulp aqueous extract (AMPW) and stored at −20°C
  3. Methanol extract of A. muricata seeds: 20 g of dried seed powder was dissolved in 100 ml methanol and extracted at 50°C for 4 h using a Soxhlet apparatus. The mixture was cooled and filtered using Whatman filter paper No. 1 and was concentrated using a rotavapor and labeled A. muricata seed methanol extract (AMSM) [Figure 2].
Figure 2: Schematic representation of phytochemical extraction procedure. To isolate phytochemicals, Annona muricata pulp and seeds were subjected to extraction using methanol and water. Experimentally, first, the Annona muricata fruit was separated in to pulp (68.72% yield) and seeds (7.29%). The skin portion contributed to the remaining (~24%) weight. The isolated pulp was freeze-dried and extracted with (a) methanol in a Soxhlet apparatus (labeled as AMPM) and (b) water (labeled as AMPW) by stirring at room temperature. Seeds were powdered and extracted with methanol (labeled as AMSM) in a Soxhlet apparatus. Yield of the AMPW was difficult to measure as we could not achieve complete drying of this fraction. AMPW = Annona muricata pulp aqueous extract, AMPM = Annona muricata pulp methanol extract, AMSM = Annona muricata seed methanol extract

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Phytochemical screening

About 10 mg/ml of AMPM was prepared in methanol and AMPW was subjected to various qualitative tests. The extracts were then subjected to phytochemical screening as follows:

  1. Dragendorff's test for alkaloids: 0.2 ml extract was taken in a test tube and added 0.2 ml dilute HCl. To this, 2–3 drops of Dragendorff's reagent was added. Appearance of orange or red precipitate and turbid solution indicates the presence of alkaloids
  2. Molisch's test for carbohydrates: 0.2 ml extract was mixed with few drops of Molisch's reagent (α-naphthol dissolved in alcohol). To this, 0.2 ml concentrated H2 SO 4 was added along the sides of the test tube. Appearance of a purple color ring at the juncture of the liquids indicates the presence of carbohydrates in the sample
  3. Braymer's test for tannins: 0.2 ml extract was mixed with 2.0 ml water and kept on water bath for 10 min. The mixture was filtered and 10% ferric chloride (FeCl3) solution was added to the filtrate. Appearance of a transient greenish to black color indicates the presence of tannin
  4. Salkowski's test for terpenoids: 0.2 ml of plant extract was mixed with 0.2 ml of CHCl3. To this, 0.2 ml of concentrated H2 SO 4 was added along the walls of the test tube. Appearance of reddish-brown color at the interface confirms the presence of terpenoids
  5. Concentrated H2 SO 4 test for glycosides: 0.2 ml of sample was mixed with 0.2 ml of CHCl3. To this, 0.2 ml of acetic acid was added and the mixture was cooled on ice. H2 SO 4 was added carefully and a change in color from violet to blue to green indicates the presence of glycosides
  6. Liebermann-Burchard test for steroids: 0.2 ml sample was mixed with 0.2 ml CHCl3. To this, 1–2 ml of acetic anhydride and two drops of concentrated H2 SO 4 was added along the sides of the test tube. Initial appearance of red, eventually blue, and finally green color indicate the presence of steroids
  7. Foam test for saponins: To 0.5 g extract, 5.0 ml distilled water was added. The solution was shaken vigorously and observed for a stable persistent froth. The frothing was mixed with three drops of olive oil and shaken vigorously and observed for the formation of an emulsion
  8. Alkaline reagent test for flavonoids: 0.2 ml plant extract was taken in a test tube and mixed thoroughly with 2 ml of 10% NaOH. To this, a few drops of diluted HCl was added. Transformation of yellow to colorless solution indicates the presence of flavonoids
  9. Folin–Ciocalteu test for phenols: To 0.2 ml extract, 0.4 ml 50% Folin–Ciocalteu reagent was added, followed by the addition of a few drops of 10% Na2 CO 3 solution. Formation of blue or green color, after 30 min incubation at room temperature, indicates the presence of phenols
  10. Keller–Killani test for cardiac glycosides: 0.2 ml extract was mixed with 10 μl of glacial acetic acid containing one drop of 5% FeCl3 solution followed by addition of 40 μl concentrated H2 SO 4. Appearance of reddish layer at the interphase, with the upper liquid layer appearing bluish-green, confirms the presence of glycosides
  11. Ruthenium test for mucilage: 0.2 ml of extract was taken on a slide containing ruthenium red solution. Development of pink coloration indicates the presence of mucilage
  12. Test for volatile oils: To 0.4 ml of extract, 0.4 ml of Sudan III TS dye was added. Presence of volatile oils will result in orange-red, red, or purplish-red color.


Antioxidant and anti-inflammatory assay

Determination of ferric reducing antioxidant power

Ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain.[62] Briefly, FRAP reagent, containing 2.5 ml of 10 mM 2, 4, 6-trypyridyl-s-triazine (heated at 50°C for 5 min), 2.5 ml of 20 mM FeCl3 solution, and 25 ml of 300 mM acetate buffer, pH 3.6, was prepared freshly and the reagent mixture warmed at 37°C just before use. A volume of 190 μl FRAP reagent was incubated with AMPM and AMSM derivatives with increasing concentrations of 25, 50, 100, and 200 μg/ml and AMPW of dilutions 0.75%, 1.56%, 3.125%, 6.25%, 12.5%, 25%, and 50%, along with ferrous sulfate (standard: 200–1800 μM) in dark for 30 min and absorbance of developed blue color measured at 593 nm. The absorbance values were converted to FRAP units (equivalent amount of ferrous sulfate) using ferrous sulfate calibration curve and the data were plotted against the concentration of compounds.

Estimation of DPPH radical scavenging activity

DPPH radical scavenging activity was measured according to Aksoy et al.[63] Briefly, 140 μl of DPPH solution (6.2 mg in 100 ml of 100% ethanol) was incubated with 20.0 μl of increasing concentrations (25, 50, 100, and 200 μg/ml) of AMPM and AMPW derivatives for 30 min in dark at room temperature and the absorbance measured at 536 nm. For construction of a calibration curve, increasing concentrations of Vitamin C (0.25–4 μM) was incubated with 140 μl of DPPH solution, and after 30 min, the absorbance was read at 536 nm. The results were expressed as % free radical scavenging activity as shown below.

Percentage free radical scavenging activity = (A0 – A1/A0) × 100

Where A0 = Absorbance of DPPH incubated with solvent and A1 = Absorbance of DPPH incubated with increasing concentration of Vitamin C/test samples.

Determination of anti-inflammatory property by RBC membrane stabilization

RBC membrane stabilization assay was performed according to Anosike et al.[64] Experimentally, first, human RBCs were pelleted by centrifuging 5.0 ml of blood and collected from the blood bank of JSS Academy of Higher Education and Research, at 2000 rpm at 4°C for 5 min. The RBC pellet was washed twice with 5 ml of iso-saline (0.9% NaCl) and resuspended in iso-saline to produce a 10% RBC suspension. Next, 500 μL of 10% RBC suspension was mixed with the test samples, 25–200 μg/ml of AMPM and AMSM, 0.75%–50% dilutions of AMPW, and 1 ml of phosphate buffer (0.15 M, pH 7.4). This reaction mixture was incubated at 37°C for 1 h. The samples were then centrifuged to collect the supernatant and the absorbance read at 560 nm in a ultraviolet-visible spectrophotometer. The RBC suspension incubated with distilled water was taken as control for complete lysis, while aspirin (1 mg/ml) served as positive control. The percentage RBCs protection compared to just water-treated control was measured and the data from three independent experiments plotted were against compound concentration.

Percentage inhibition of RBC lysis = (A0 – A1/A0) × 100

Where A0 = Absorbance of 10% RBC suspension incubated with distilled water and A1 = Absorbance of 10% RBC suspension incubated with test samples.

Anticancer assay

Cytotoxicity assay

The cytotoxic effects of the extracts were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, commonly known as MTT.[65] Breast cancer cell lines MDA MB-231 and MCF-7 were procured from ATCC, cultured in DMEM supplemented with 10% fetal bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 μg/ml) in 5% CO2, at 37°C, until confluent. The cells were trypsinized using 0.25% trypsin-EDTA solution and viability assessed. 100 μl of the media-diluted cell suspensions, containing 10,000 cells/well, was plated and incubated in 5% CO2 at 37°C, till confluence. The cells were treated with 10, 20, 40, 80, 160 and 320 μg/ml concentrations of AMPM and AMSM or 0.75%, 1.56%, 3.125%, 6.25%, 12.5%, 25%, and 50% dilutions of AMPW.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

MTT assay was performed according to Denizot and Lang.[65] After 24 h of treatment, cells were incubated with MTT reagent (5 mg/ml) at 37°C for 1 h and centrifuged at 3000 rpm for 5 min. Plates were removed from centrifuge and excess dye was washed and 100 μl of DMSO was added to solubilize the formazan crystals. Absorbance was measured at 570 nm on a multimode plate reader. The percentage inhibition was calculated using the below formula:

%inhibition = [(OD of control -OD of sample)/OD of control]x100

Apoptosis detection by flow cytometry

Apoptosis detection was performed using propidium iodide (PI) and annexin V-fluorescein isothiocyanate (AV-FITC), according to Vermes et al.[66] In brief, 0.5 × 106 MDA-MB-231 and MCF-7 cells/well were plated in two different six-well plates using DMEM supplemented with 10% FBS and 1% penicillin-streptomycin, incubated overnight at 37°C at 5% CO2. The media were discarded and the cells were treated with increasing concentrations of AMPM and AMPW (prepared using media containing 10% FBS). After 24 h incubation, the cells were harvested; contents were transferred to sterile FACS tubes and centrifuged at 2000 rpm for 5 min. The supernatant was discarded and the cells were washed with cold phosphate-buffered saline (PBS) and re-suspended in 1 ml 1X binding buffer. 500 μl of cell suspension (~5 × 105 cells) was aliquoted to a new FACS tube and added with 5 μl of annexin-V and 10 μl of PI, followed by a gentle mixing and incubation for 15 min in dark at room temperature. The cells were analyzed by flow cytometry within 1 h postincubation.

Cell cycle analysis by flow cytometry

Experimentally, 32 mg of AMPM was dissolved in 1 ml of DMSO (filtered), as a stock, and further diluted to 160 and 320 μg/ml using complete media, while 100% concentration of AMPW was diluted directly to 12.5% and 25% concentrations using complete media. Distribution of cells in different phages of cell cycle was measured according to Azimian-Zavareh et al.[67] Experimentally, 0.5 × 106 MDA-MB-231 and MCF-7 cells/well were seeded and cultured, using 2.0 ml serum-free DMEM, in two different six-well plates. After 24 h, desired concentrations of the extracts, prepared in complete media, were added and the treatment continued for 24 h. The treated cells were then harvested and centrifuged at 2000 rpm. The cell pellet was washed twice by re-suspending in 2.0 ml of 1X PBS and fixed using 300 μl of sheath fluid. This was then followed by drop-by-drop addition of 1 ml of chilled 70% ethanol, while mixing gently, and another 1 ml of chilled 70% ethanol was added at once. The cells were then stored at 4°C overnight for fixing. Postfixing, the cells were centrifuged for 5 min at 2000 rpm and the cell pellet was washed twice with 2 ml of cold 1X PBS. Following which, the cells were re-suspended in 500 μl of sheath fluid supplemented with 0.05 mg/ml of PI and 0.05 mg/ml of RNase A and incubated in dark for 15 min. The percentage cells in various cell cycle stages, in both control and treated populations, were determined using FACSCalibur.

Statistical analysis

All the experiments were conducted in triplicates and the results were expressed as mean ± standard error of the mean (SEM) until or otherwise stated. The data were compiled into the Prism 8 statistical analysis tool, by GraphPad Software, San Diego, CA, and were subjected to non-parametric unpaired t-test to determine the difference between A. muricata extracts and controls/positive controls. P < 0.05 was considered statistically significant.


 > Results Top


Extraction and phytochemical screening

The total yield of phytochemicals obtained from 60 g of A. muricata pulp, extracted using methanol, was 7.97 g (13.28%). Phytochemical screening indicated the presence of phenols, steroids, tannins, flavonoids, terpenoids, cardiac glycosides, alkaloids, carbohydrates, and protein as the constituent phytochemicals of AMPM [Table 1]. AMPW was tested positive for the presence of saponins, phenols, steroids, tannins, terpenoids, alkaloids, carbohydrates, and protein. No traces of glycosides, mucilage, and volatile oils were detected in A. muricata pulp extracts. Compared to pulp, the methanolic extract of seed yielded less (~8.21%) material [Table 1].
Table 1: Qualitative analysis of methanolic and aqueous extracts of Annona muricata pulp. Qualitative analysis of methanolic and aqueous extracts of Annona muricata pulp showed the presence of phenols, steroids, tannins, terpenoids, alkaloids, and carbohydrates in both extracts. However, flavonoids, cardiac glycosides and glycosides were absent in AMPW. Likewise, the AMPM is devoid of saponins and glycosides

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Antioxidant and anti-inflammatory properties of Annona muricata pulp methanol extract, Annona muricata seed methanol extract, and Annona muricata pulp aqueous extract

Antioxidant activity potential of AMPM and AMPW was determined using FRAP [Supplementary Figure 1]; and [Figure 3]a and [Figure 3]b and DPPH free radical scavenging activity [Supplementary Figure 2]; and [Figure 4]a and [Figure 4]b assays. Experimentally, increasing concentrations of AMPM, AMSM, and AMPW were incubated with DPPH/FRAP reagents as detailed in “Methods” Section, and the developed color was measured at 536 and 593, nm, respectively, using a multimode plate reader. The data demonstrated a dose-dependent increase in free radical scavenging activity of the extracts. In addition to free radical scavenging activity, the A. muricata extracts possessed potent FRAP. However, AMPM and AMPW exhibited very low antioxidant activity at concentrations <20 μg/ml.

Figure 3: FRAPs of AMPM, AMPW, and AMSM. To determine the FRAP of Annona muricata extracts, increasing concentration of samples was incubated with FRAP reagent as detailed in methods section and number of FRAP units determined. Analysis of the data showed a dose-dependent increase in antioxidant activity by AMPM, AMPW and AMSM. Both AMPM and AMSM showed similar antioxidant potential (a). AMPW exhibited a significant decrease in antioxidant potential with increasing dilution of the fraction (b). Data represent the mean values with standard error of mean of three independent experiments with at least three replicate measurements in each experiment. AMPW = Annona muricata pulp aqueous extract, AMPM = Annona muricata pulp methanol extract, AMSM = Annona muricata seed methanol extract, FRAP = Ferric reducing antioxidant potential

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Figure 4: Free radical scavenging potentials of AMPM, AMPW and AMSM. Since the Annona muricata extracts exhibited potent FRAP, next, the free radical scavenging potential was determined using DPPH method. Extracts AMPM (a) and AMPW (b) showed a dose-dependent increase in free radical scavenging potential. Interestingly, the AMSM exhibited very high DPPH radical scavenging potential (~80%) even at extract the lowest dose (10 μg/ml) tested (a). Compared to AMPW, the radical scavenging activity of AMPM and AMSM is much higher. Data represent the mean values with standard error of mean of three independent experiments with at least three replicate measurements in each experiment. AMPW = Annona muricata pulp aqueous extract, AMPM = Annona muricata pulp methanol extract, AMSM = Annona muricata seed methanol extract, FRAP = Ferric reducing antioxidant potential

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Antioxidants are known to exhibit potent RBC membrane stabilizing effect.[64] Since RBC membranes structurally resemble the membranes of lysosomes, it is important to study the effect of phytochemical extracts on RBC membrane stabilization. Experimentally, the anti-inflammatory effect was carried out by incubating increasing concentrations of AMPM, AMSM, and AMPW with the RBC followed by measuring the hemoglobin content in the medium [Figure 5]a and [Figure 5]b. It is hypothesized that potent antioxidants protect RBC from undergoing membrane damage. Analysis of the data showed that AMPW exhibited the highest anti-inflammatory property compared to other extracts. Nonetheless, all the test samples demonstrated a dose-dependent anti-inflammatory activity in vitro .
Figure 5: RBC membrane stabilization potentials of AMPM, AMPW and AMSM. RBC membrane stabilization is an indicator of the potential of antioxidants to protect cells from lysosomal degradation. In order to determine the RBC membrane stabilization potential, the extracts were added to the RBC and the ability to protect from heat induced lysis assessed as described in methods. AMPM, AMSM (a) and AMPW (b), showed a dose dependant increase in RBC membrane protection. Higher doses of 320g/ml AMPM and 50% AMPW where found to be more potent in stabilizing RBC membrane as compared to 100g/ml Aspirin positive control. AMPW showed best protect compared to AMPM and AMSM. Data represents the mean values with standard error of mean of three independent experiments with at least three replicate measurements in each experiment. AMPW = Annona muricata pulp aqueous extract, AMPM = Annona muricata pulp methanol extract, AMSM = Annona muricata seed methanol extract

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Anticancer effect of Annona muricata pulp methanol extract, Annona muricata seed methanol extract, and Annona muricata pulp aqueous extract

Methanol extracts of A. muricata pulp and seed and aqueous extract of A. muricata pulp were tested for determining the potency against cancer cell lines representing breast. Dose–response study conducted for 24 h using MCF-7 and MDA-MB-231 cell lines treated with 10–320 μg/ml AMPM and AMSM and 0.75%–50% AMPW showed an about 42% growth inhibition at 320 μg/ml with AMPM [Figure 6], [Figure 7], [Figure 8].
Figure 6: Effect of AMPM on MCF-7 and MDA-MB-231 cells survival: To determine the cytotoxic potential of AMPM, MCF-7 and MDA-MB-231 cells were plated and exposed to increasing concentration of AMPM (weight/volume). (a) Number of viable cells was determined using MTT assay. AMPM exhibited a dose-dependent increase in the % inhibition of MCF-7 cells. At 320g/ml, AMPM inhibited 41.7% of tumor cells. The positive control, 50M Cisplatin, yielded ~52.4% inhibition. (b) Similarly, exposure of MDA-MB-231 cells to AMPM retarded the growth by 40.89%. The positive control Cisplatin yielded about 59.184% inhibition. Analysis of the photomicrographs showed a rounded morphology of cells upon treatment with AMPM (c and d). In addition, the number of cells was much lesser in the AMPM-treated group. n = 3, mean ± SEM, P < 0.05 was considered statistically significant,*P < 0.05, **P < 0.01, ***P < 0.0001, and ns = Not significant. AMPM = Annona muricata pulp methanol extract, SEM = Standard error of the mean

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Figure 7: Effect of AMPW on MCF-7 and MDA-MB-231 cells survival: To determine the cytotoxic potential of water extract of Annona muricata pulp (AMPM), MCF-7 and MDA-MB-231 cells were plated and exposed to AMPW (volume/volume). Number of viable cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. AMPW exhibited a dose-dependent increase in the % inhibition of MCF-7 cells. At 50% dilution, AMPW inhibited ~82% of tumor cells. The positive control, 50 μM cisplatin, yielded ~52.4% inhibition (a). Similarly, exposure of MDA-MB-231 cells to AMPW retarded the growth by ~89.0%. The positive control cisplatin yielded about 59.184% inhibition (b). Analysis of the photomicrographs showed a rounded morphology of cells upon treatment with AMPM (c and d). In addition, the number of cells was much lesser in the AMPM-treated group. n = 3, mean ± SEM, P < 0.05 was considered statistically significant,*P < 0.05, **P < 0.01, ***P = <0.0001, and ns = Not significant. AMPM = Annona muricata pulp methanol extract, AMPW = Annona muricata pulp aqueous extract, SEM = Standard error of the mean

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Figure 8: Effect of AMSM on MCF-7 and MDA-MB-231 cells survival: To determine the cytotoxic potential of methanolic extract of A. muricata seed (AMSM), MCF-7 and MDA-MB-231 cells were plated and exposed to increasing concentration of AMSM (weight/volume). Number of viable cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. AMSM exhibited a dose-dependent increase in the %inhibition of MCF-7 cells. 320 μg/ml of AMSM inhibited ~82% of tumor cells. The positive control 50 μM cisplatin yielded ~52.4% inhibition (a) Similarly, exposure of MDA-MB-231 cells to AMSM retarded the growth by ~80%. The positive control cisplatin yielded about 59.184% inhibition (b). Analysis of the photomicrographs showed a rounded morphology of cells upon treatment with AMSM (c and d). In addition, the number of cells was much lesser in the AMSM treated group. n = 3, mean ± SEM, P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.0001, and ns = Not significant. AMSM = Annona muricata seed methanol extract, SEM = Standard error of the mean

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Similarly, 24 h treatment with 320 μg/ml AMSM inhibited the growth of MCF-7 cells by 83.98% and MDA-MB-231 cells by 81.35% with IC50 of 140.2 and 153.5 μg/ml, respectively, for MCF-7 and MDA-MB-231. Analysis of the photomicrographs showed change in the cell morphology and cell death at concentrations higher than 160 μg/ml.

Apoptosis inducing potential of Annona muricata pulp methanol extract, Annona muricata seed methanol extract, and Annona muricata pulp aqueous extract

In an attempt to comprehend the inhibition of cell proliferation, in both the MCF-7 and MDA-MB-231 cells, and apoptosis, flow cytometric detection was carried out by staining the untreated [Figure 9]a and [Figure 9]d; [Figure 10]a and [Figure 10]d and treated cells and treated cells with PI and AV-FITC using BD-FACSCalibur. The analysis demonstrated a dose-dependent increase in the percentage of cells under early and late phase apoptosis as well as necrosis.
Figure 9: AMPM-induced apoptosis in MCF-7 and MDA-MB-231 cells: To determine the processes modulated by AMPM treatment, first, apoptosis level was quantified using FACS as detailed in methods section. (a and d) The scattering of untreated MCF-7 and MDA-MB-231 cells. About 99.20% and 96.66% cells are viable, respectively, in MCF-7 and MDA-MB-231. (b and e) Plots of MCF-7 treated with 160 μg/ml of AMPM. An about 16.10% cells were found in the necrotic region. However, ~0.60% cells were located in late apoptotic and 0.12% in early apoptotic regions. About 83.18% cells were observed in viable region of the plots. Similarly, MDA-MB-231 cells showed 26.36%, 13.56%, 7.24% and 52.84% respectively, necrotic, late apoptotic, early apoptotic and viable cells. (c and f) Increasing the concentration of AMPM to 320 μg/ml showed 68.62% and 38.60% viable cells in MCF-7 and MDA-MB-231 cell lines. However, the AMPM treatment produced 0.04% and 2.12%; 0.38% and 10.42%; and 30.96% and 50.86%, respectively of early apoptosis, late apoptosis, and necrosis, respectively, in MCF-7 and MDA-MB-231 cells, respectively, treated for 24 h. Data represent the mean values with standard error of mean of three independent experiments with at least three replicate measurements in each experiment. AMPM = Annona muricata pulp methanol extract

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Figure 10: AMPW-induced apoptosis in MCF-7 and MDA-MB-231 cells: In order to determine the processes modulated by AMPW treatment, first, apoptosis level was quantified using FACS as detailed in “Methods” Section. (a) and d) The scattering of untreated MCF-7 and MDA-MB-231 cells. About 99.20% and 96.66% cells are viable, respectively, in MCF-7 and MDA-MB-231. (b and e) Plots of MCF-7 treated with 12.5% of AMPW. An about 24.56% cells were found in the necrotic region. However, ~0.18% cells were located in late apoptotic and 0.06% in early apoptotic regions. About 75.20% cells were observed in viable region of the plots. Similarly, MDA-MB-231 cells showed 37.98%, 0.04%, 0.10%, and 61.88%, respectively, necrotic, late apoptotic, early apoptotic, and viable cells. (10c and f) Increasing the concentration of AMPW to 25 μg/ml showed 35.50% and 40.66% viable cells in MCF-7 and MDA-MB-231 cell lines. However, the AMPW treatment produced 1.30% and 0.14%; 10.08% and 0.06%; and 53.12% and 59.14%, respectively of early apoptosis, late apoptosis, and necrosis, respectively, in MCF-7 and MDA-MB-231 cells, respectively, treated for 24 h. Data represent the mean values with standard error of mean of three independent experiments with at least three replicate measurements in each experiment. AMPW = Annona muricata pulp aqueous extract

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MCF-7 cells treated with 160 μg/ml of AMPM for 24 h showed 0.12% of early apoptotic, 0.60% of late apoptotic, and 16.10% of necrotic cells. However, when the concentration increased to 320 μg/ml, the cells displayed 30.96% necrosis, 0.38% late apoptosis, and 0.04% early apoptosis [Figure 9]b and [Figure 9]c. Treatment of MDA-MB-231 cells for 24 h with 160 and 320 μg/ml AMPM showed a reduction in the number of viable cells. For instance, 26.36% and 50.86% necrotic, 13.56% and 10.42% late apoptotic, and 7.24% and 2.12% early apoptotic cells were observed in the 160 μg/ml and 320 μg/ml treatments [Figure 9]e and [Figure 9]f, respectively, indicating a greater efficacy of AMPM over MDA-MB-231 compared to MCF-7.

Twenty-four-hour treatment of MCF-7 and MDA-MB-231 cells with 25% AMPW showed 0.06% and 0.14% of early apoptosis, 0.18% and 0.06% of late apoptosis and 24.56% and 59.14% of necrosis, respectively. Doubling the concentration of the extract increased the necrotic population to 53.12%, late apoptotic cells to 10.08%, and early apoptotic cells to 1.30% in MCF-7 cells [Figure 10]b and [Figure 10]c. Similarly, a significant increase was observed in MDA-MB-231 cells (59.14% necrosis, 0.06% late apoptosis, and 0.14% early apoptosis) [Figure 10]e and [Figure 10]f.

Cell cycle arrest by Annona muricata pulp methanol extract, Annona muricata seed methanol extract, and Annona muricata pulp aqueous extract

To understand the cell growth inhibition mechanisms, cell cycle analysis was carried out. The untreated and A. muricata pulp extracts treated MCF-7 and MDA-MB-231 cells were stained with PI and analyzed using BD-FACSCalibur as explained in “Methods” Section.

An increase in the G0/G1 phase population was observed in both MCF-7 as well as MDA-MB-231 cells were exposed to both 160μg/ml and 320μg/ml concentrations of AMPM [Figure 11] and 12.5% AMPW [Figure 12], respectively, for 24h. Additionally, both the groups, treated with AMPM and AMPW, demonstrated increased S-phase and decreased G2/M phase populations, as compared to the positive control treated group. The positive control 50μM cisplatin arrested MCF-7 as well as MDA-MB-231 cells in G2/M phase. Unlike MDA-MB-231, the MCF-7 cells treated with both the extracts showed an increase in sub-G0-G1 and S-phase population at 12.5% AMPW treatment. The positive control 50μM cisplatin arrested MCF-7 cells in G2/M phase. At higher concentrations, the values observed were not reproducible for AMPW.
Figure 11: AMPM-induced G0/G1 cell cycle arrest and sub-G0/G1 cell population in MCF-7 and MDA-MB-231 cells: To determine the effect of AMPM on cell cycle progression, MCF-7 (a and c) and MDA-MB-231 (b and d) cells were exposed to 160 and 320 μg/ml AMPM, and positive control 50 μM cisplatin for 24 h and the control and treated cells stained using propidium iodide as detailed in methods section. Analysis of the data showed an increased sub-G0-G1 and G0/G1 cell population upon treatment with 160 μg/mL AMPM. (b and d) Effect of AMPM on MDA-MB-231 cells treated with 160 and 320 μg/ml of AMPM and cisplatin. Similar to MCF-7, a significant increase in G0/G1 cell population was observed at 160 μg/ml AMPM. However, interestingly, no sub-G0-G1 population was observed in this cell line. Positive control cisplatin-induced G2/M cell cycle arrest in both cell lines. AMPM = Annona muricata pulp methanol extract

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Figure 12: AMPW modulates cell cycle stages differentially in MCF-7 and MDA-MB-231 cells: To determine the effect of AMPW on cell cycle progression, MCF-7 (a and c) and MDA-MB-231 (b and d) cells were exposed to 12.5% AMPW and positive control 50 μM cisplatin for 24 h, and the control and treated cells stained using propidium iodide as detailed in “Methods” Section. Analysis of the data showed an increase in sub-G0-G1 and S-phase population only in MCF-7. (b and d) The effect of AMPW on. However, in MDA-MB-231, AMPW induced G0/G1 cell cycle arrest. Minor changes were also observed in S-phase and G2/M population in MDA-MB-231 cells. Positive control cisplatin induced G2/M cell cycle arrest in both cell lines. The values were not reproducible in higher concentrations. AMPW = Annona muricata pulp aqueous extract

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 > Discussion Top


Currently, treatment options for breast adenocarcinoma are still bleak owing to (a) rapid evolution of drug resistant forms; (b) minimal success with existing therapeutics; and (c) systemic toxicity with the nontargeted therapies.[4] Hence, the use of natural products to treat breast cancers is gaining importance in the recent years. A few such natural products have been successful in inhibiting any one or few of the several pathways that trigger tumorigenesis and metastasis of the breast cancer cells.[68],[69],[70] For instance, curcumin, derived from turmeric, is known to contribute toward cancer cell death by apoptosis via either the intrinsic or the extrinsic pathways. Intrinsically, it disturbs the balance in mitochondrial membrane potential to suppress the anti-apoptotic Bcl-xL protein. Meanwhile, increase in DR4 and DR5 death receptors population on the cell surface and trigger of tumor necrosis factor-mediated apoptosis are believed to occur via extrinsic pathway, upon treatment with curcumin.[71] In another study, involving treatment of BALB/c nude mice-bearing MDA-MB-453 cell xenografts, oral administration of anthocyanin-rich extract from black rice showed significant suppression of angiogenesis along with tumor reduction. Further investigation suggested downregulation of MMP9, MMP2, and uPA expression in the tumor tissues was the mechanistic cause behind the above growth regulation.[72]

Likewise, various parts of the A. muricata plant have been frequently investigated as cancer therapeutics,[73] and different solvent extracts of A. muricata have been reported to have varied specificity in their anticancer properties, i.e., while n-hexane and chloroform solvent extracts exhibited activity against cervical cancer only, hexane, ethyl acetate, ethanol, and methanol solvent extracts demonstrated activity against a wider range of cancers.[74] Syed Najmuddin et al. reported that the water extracts of A. muricata leaves were not just cytotoxic against breast cancer cell lines MDA-MB-231, MCF-7, and 4T1 but also proved to be effective anti-inflammatory agents by reducing nitric oxide and malondialdehyde levels and increasing the number of T-lymphocytes and NK-cells in vivo.[75] Moreover, A. muricata phytochemicals extracted using methanol were also found to be effective against multi-factorial drug-resistant cell lines, CEM/ADR5000 leukemia cells.[76] Wide range of bioactivity and presence of unique phytochemicals in the plant have invoked interest and attention of researchers worldwide. In a study by Ko et al., observations suggested induction of apoptosis, by A. muricata phytochemicals, via Endoplasmic Reticulum-mediated pathway.[77] Similarly, AGEs were reported to inhibit NADH-oxidase and mitochondrial complex 1, thereby resulting in apoptosis due to lower ATP levels.[78] In vivo prevention of DNA damage and corresponding protection against development of DMBA-induced breast cancer by the A. muricata leaves extract can be considered promising.[79] A case study by Hansra et al. reported that consumption of 8oz. of A. muricata leaves decoction, once every day, helped in stabilizing a metastatic breast cancer for as long as 5 years.[80]

Notwithstanding, studies pertaining to the A. muricata fruit, the edible as well as most commonly consumed part of the plant, have been negligible. Therefore, the current study explored and compared the efficacy of fruit pulp and seeds, for inhibiting Estrogen-receptor and progesterone-recetor (ER- and PR)-positive MCF-7 cell line and ER- and PR-negative cell line MDA-MB-231. Preliminary antioxidant and anti-inflammatory studies conducted for all the three samples, AMPM, AMSM, and AMPW, indicated the presence of phytochemicals with possible anticancer potential. Consistently, AMPM, AMPW, and AMSM reduced the viability of MCF-7 as well as MDA-MB-231 breast cancer cell lines by inducing necrosis and cell cycle arrest. The cytotoxicity assays revealed that the phytocompounds present in all the extracts, AMPM, AMPW, and AMSM, induced cell death. While the AMSM showed the highest growth inhibition, the AMPM showed least cytotoxicity. However, since the A. muricata fruit pulp is more commonly consumed as compared to its seeds, AMSM was excluded for further studies and cytotoxicity of the fruit pulp methanol and water extracts, i.e., AMPM and AMPW, was subjected to cytometric analysis. The cytometric observations strongly endorsed the pro-apoptotic compulsions of G0/G1 phase cell cycle arrest responsible for the observed cytotoxicity.[77] Flow cytometry analysis demonstrated that AMPW exhibited much better tumoricidal activity compared to AMPM. However, both the fractions were on par with the cisplatin-positive control in causing the cell cycle arrest and cell death. Since the yield of phytochemicals was unknown in the case of AMPW, the AMPM was found to be a more suited option for further exploration of antibreast cancer property of A. muricata.


 > Conclusion Top


The observations made in the in vitro assays showed the antioxidant, anti-inflammatory, and anticancer potentials of AMPM, AMPW, and AMSM. Dose-dependent antioxidant, free radical scavenging, anti-inflammatory properties as well as the retardation of cell proliferation were observed when MCF-7 and MDA-MB-231 cell lines were exposed to the extracts for 24 h. Mechanistically, the extracts have arrested the cells in G0/G1 phase and induced death through promoting necrosis. Further studies pertaining to detailed pathway analyses and identification of phytochemicals responsible for the tumoricidal activity and toxicity of the extracts are essential for developing a formulation for further testing.

Contributions to the field

Breast cancer is one of the leading causes of death due to cancers in women globally. Despite numerous studies, to date, no clinically viable therapeutic agent is available to treat advanced tumors. In addition, development of treatment-resistant phenotypes necessitates the need for combination therapies. However, a clinically successful combination regimen is not available to date. Hence, identification of a plant-based tumoricidal fraction is probably a viable option to treat breast cancers. The current study aimed at developing a potent phytochemical fraction from A. muricata, a miracle plant with known knowledge about its potency for treating various health complications. Therefore, the cytotoxic potential of A. muricata pulp has been tested for antibreast cancer activity and the processes responsible for treatment-induced death were identified. Results of this study help in developing a potent nutraceutical formulation to treat breast cancers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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