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Syzygium cumini (jamun) fruit-extracted phytochemicals exert anti-proliferative effect on ovarian cancer cells


1 Department of Gynecology, Jinan Municipal Hospital of Traditional Chinese Medicine, Jinan, Shandong Province, China
2 Department of Pharmacy, Birla Institute of Technology and Sciences, Hyderabad Campus, Hyderabad, Telangana, India
3 Division of Biochemistry, Faculty of Basic Sciences, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, Jammu, India
4 Centre for Molecular Biology, Central University of Jammu, Samba, Jammu and Kashmir, India

Date of Submission22-Feb-2020
Date of Decision30-May-2020
Date of Acceptance12-Aug-2020
Date of Web Publication30-Jul-2021

Correspondence Address:
Audesh Bhat,
Centre for Molecular Biology, Central University of Jammu, Rahya-Suchani (Bagla), Samba -181 143, Jammu and Kashmir
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_210_20

 > Abstract 


Background: The medicinal properties of Syzygium sp., especially the antidiabetic property, date back to the ancient times. However, in the recent past, extracts from different parts of the Syzygium sp. have demonstrated promising anticancer activities in diverse cancer types, and now, attempts are being made to identify the active phytochemicals.
Aims and Objectives: In this study, we intended to test the anticancer properties of phytochemicals extracted from the fruit of Syzygium cumini plant in ovarian cancer cells.
Materials and Methods: A total of nine phytochemicals extracted from the S. cumini fruits using chloroform were tested for their anticancer activity in the ovarian cancer cell line PA-1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium assay was performed to calculate the 50% inhibition (IC50) concentration and cell cytotoxicity values. Cell scratch assay was performed to assess the proliferation inhibition activity of the phytochemicals. Cisplatin was used as positive control.
Results: Out of the nine phytochemicals tested, quercetin (QC), gallic acid (GA), and oleanolic acid (OA) were found active. QC and GA were most effective with more than 90% cell cytotoxicity at 2.5 µ g/ml and above concentrations and OA moderately effective up to 5 µg/ml serial concentrations. Cell proliferation was significantly inhibited by QC and GA and moderately but significantly by OA.
Conclusion: Our data demonstrate the anticancer activity of QC, GA, and OA phytochemicals, which is consistent with the previous reports. However, this is the first report showing the anticancer activity of these phytochemicals derived from S. cumini in the ovarian cancer cells. These data suggest that there is a potential to develop these phytochemicals as anticancer therapeutic agents either as monotherapeutic agents or in combination with commonly used chemotherapeutic agents, which needs to be explored.

Keywords: Anticancer activity, cell proliferation, chemotherapy, cytotoxicity, ovarian cancer, phytochemicals, Syzygium cumini



How to cite this URL:
Li L, Mangali S, Kour N, Dasari D, Ghatage T, Sharma V, Dhar A, Bhat A. Syzygium cumini (jamun) fruit-extracted phytochemicals exert anti-proliferative effect on ovarian cancer cells. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322708




 > Introduction Top


Plants have been the source of medicine since the ancient times and continue to be vital components of the traditional healthcare system of several countries, especially India and China. Decades of research has led to the identification of several plant-based chemicals, commonly known as phytochemicals with promising chemopreventive and chemotherapeutic properties, thus having potential to be used in the development of new anticancer drugs.[1] Phytochemicals are biologically active nonnutritive chemicals such as carotenoids, flavonoids, alkaloids, polyphenols, and isoflavones of plant origin possessing many health benefits.[2] With more than 5000 already identified and many more yet to be identified, phytochemicals are emerging as promising resource of natural compounds with health benefits and as an alternative to synthetic drugs.[2],[3] Besides cancer, phytochemicals have shown preventive and treatment properties against diabetes, heart disease, hypertension, and many other medical conditions.[3],[4] The importance of phytochemicals in cancer treatment is of particular importance because majority of the conventional chemotherapeutic agents, although in wide use since the mid-20th century owing to their high efficacy,[5] have adverse side effects and have reached an efficacy plateau against most solid tumors with majority of the patients eventually developing chemoresistance.[6],[7],[8],[9] For these reasons, treatment with conventional chemotherapeutic agents often results in poor treatment outcomes, thus highlighting the need for development of better and tolerable therapeutic agents.[8],[10] Being toxic only to cancer cells but not to normal cells and having no side effects, phytochemicals have gained wide acceptance in recent times to treat cancer with many phytochemicals such as curcumin, genistein, resveratrol, and sulforaphane currently undergoing clinical trials.[11] In addition, phytochemicals are also being tested in combination with conventional anticancer chemotherapeutic agents to minimize the side effects and improve prognosis.[10]

Syzygium cumini, commonly known as black plum, jamun, jambolana, Indian blackberry, Java plum, or Malabar plum, is a tropical/subtropical tree with a wide range of medicinal properties, importantly antidiabetic, anticancer, and antimicrobial activities.[12],[13],[14],[15] The use of S. cumini in traditional medicine such as Ayurveda, Homeopathy, and Unani medicine to treat diabetes and many other diseases is well known.[13],[16],[17] Besides this, phytochemical extracts or the whole extracts derived from different parts of Syzygium species have radioprotective and cancer preventive properties.[1],[15] In the present study, we investigated the anticancer properties of nine compounds extracted from S. cumini fruit in the in vitro model of ovarian teratocarcinoma cell line PA-1.


 > Materials and Methods Top


Cell culture

Ovarian teratocarcinoma cell line PA-1 and rat kidney epithelial cell line NRK-52E were obtained from National Centre for Cell Science, Pune, India. Cells were cultured and maintained in Dulbecco's Modified Eagle's Medium (HyClone, USA) containing 10% fetal bovine serum (HiMedia, India) and 1% penicillin-streptomycin (Invitrogen, USA) at 37°C in 5% CO2 incubator.

Phytochemicals and treatment

Chloroform-soluble extracts from S. cumini fruit pulp were prepared and characterized in the laboratory of Dr. Vikas Sharma from Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu, Jammu and Kashmir, India, following previously reported procedure.[18] Stock solutions of 10 mg/mL were prepared by dissolving the extracts in DMSO and were prepared at least 1 day in advance. The test chemicals were used at the final concentrations of 0.5, 1.0, 2.5, and 5 µg/ml. Cisplatin (Sigma) was used as control.


 > Cytotoxicity and 50% inhibition concentration estimation Top


Cytotoxicity of selected phytochemicals was measured using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay as described previously.[19] Briefly, PA-1 ovarian cancer cells were seeded at a density of 1 × 104 cells/well in 96-well plates and incubated overnight in humidified air with 5% CO2 at 37°C. Cells were then treated with a working concentration (0.0, 0.5, 1.0, 2.5, and 5 µg/ml) of phytochemicals for 48 h. After 48 h of incubation, MTT assay was performed using wells without cells as the blank. Noncancerous cell line NRK-52E was used as control to test the cytotoxicity of selected phytochemicals on normal cells. Cell viability was calculated according to the following formula: Cell viability (%) = cells (sample)/cells (control) ×100. Cisplatin at 5, 10, 15, 20, and 25 µM/ml was used as positive control. 50% inhibition concentration (IC50) values of the selected phytochemicals were also assessed by the MTT assay and calculated using log formula.[20],[21]

Cell migration assay

PA-1 cells (1 × 105) were seeded in six-well cell culture plate and allowed to attach overnight. Cells were treated with IC50 of selected phytochemical compounds for 48 h. Parallelly, an untreated group was used as control. After the treatment, a scratch line was made using a pipette tip. The cells were washed with phosphate-buffered saline to remove the debris; thereafter, fresh media were added and cells were allowed to grow for another 48 h. Images of the cells were taken at the place of the wound at 0 h and 48 h using phase-contrast microscope.[22],[23] ImageJ plugin MRI Wound Healing Tool (Volker Baecker, Montpellier RIO Imaging) was used to quantify cell migration into the scratch area.

Statistical analysis

GraphPad Software (GraphPad Software, San Diego California USA, www.graphpad.com) was used to perform one-way ANOVA for statistical comparison between untreated and treated samples. P < 0.05 was considered statistically significant.


 > Results Top


Out of the nine phytochemicals tested for their anticancer activity in the ovarian cancer cell line PA-1, only three phytochemicals – quercetin (QC), gallic acid (GA), and oleanolic acid (OA) – were found active. In this article, we present the data of only the active compounds. Details of other compounds will be published separately as the same compounds have been tested in other cancer types (manuscript under preparation). The IC50 values of the QC, GA, and OA were found to be 1.31, 1.73, and 3.09 µg/ml, respectively. The cell cytotoxicity values of the active compounds are given in [Figure 1]. As shown in [Figure 1], GA at 2.5 and 5.0 µg/ml concentrations was most effective with >90% cell death, followed by QC with >80% cytotoxicity at the 2.5 and 5.0 µg/ml concentrations, which were significantly higher than the DMSO-treated group (P < 0.005). OA on the other hand even though had >50% cell toxicity at the lowest concentration tested (0.5 ug/ml), however, no increase was observed after increasing the concentration” [Figure 1]. Cisplatin treatment at and above 1 µM/ml concentration resulted in >50% of cell death, consistent with the published data in PA-1 cells.[24] The treatment of NRK-52E, on the other hand, with QC and GC at the given concentrations did not result in any cell death; however, OA at 1, 2.5, and 5 µg/ml concentrations caused 25%, 37%, and 29% cell death, respectively. The cell migration data are given in [Figure 2]a and [Figure 2]b. The size of the scratch (cell-free zone) in the GA and QC treatment groups was comparable with the cisplatin group and significantly more than the control group (P < 0.005), whereas in the OA group, the area was comparably less but more than the control group (P < 0.05) [Figure 2]a. [Figure 2]b represents quantitative analysis of the scratch assay. The scratch area was calculated from three independent experiments.
Figure 1: Cytotoxicity values of the active phytochemicals extracted from the Syzygium cumini fruit. Cells were treated with the selected concentrations for 48 h. The values are mean ± SD of three separated experiments. QC = Quercetin, GA = Gallic acid, OA = Oleanolic acid, SD = Standard deviation

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Figure 2: Cell scratch assay. (a) Representative images of the scratch area in the different treatment groups at 0 and 48 h. Black bars represent the wound area. (b) Bar diagram representing the wound area calculated from three independent experiments using ImageJ program. Data represented are mean ± SD. QC = Quercetin, GA = Gallic acid, OA = Oleanolic acid, SD = Standard deviation

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 > Discussion Top


Ovarian cancer is one of the leading causes of gynecologic cancer-related deaths worldwide, with less than 40% 5-year survival rate across all stages.[25] Although ovarian cancer patients respond well to the frontline platinum-based chemotherapeutic treatment, the adverse side effects and the development of resistance to the treatment are major concerns.[26] The need to develop alternative therapeutic agents that are safe and efficacious has led to a spurt in the exploration of medicinal plants for their anticancer properties. In the present study, we investigated anticancer properties of nine chloroform-extracted phytochemicals from the S. cumini fruit and found QC, GA, and OA with significant antiproliferative activity with QC and GA showing >90% cytotoxicity even at low concentrations. The anticancer properties of several Syzygium sp.-derived compounds or whole extracts have been previously demonstrated in different cancers such as breast, liver, colon, and cervical using both in vitro and in vivo approaches as reviewed by Chua et al.[15] However, to our best knowledge, this is the first report showing anticancer activity of chloroform-extracted phytochemicals from the fruits of S. cumini in ovarian cancer cell line PA-1. Although extracts from different parts of S. cumini using different solvents have been tested for their anticancer properties, there is hardly any anticancer activity data on the S. cumini-derived pure phytochemicals. Oral administration of S. cumini fruit water extract was reported to cause significant reduction in the tumor incidence, tumor burden, and cumulative number of gastric carcinoma in benzo[a]pyrene-induced gastric cancer mouse model.[27] In another study, methanolic extract from partially ripe S. cumini fruit skin showed potent cytotoxicity in HeLa and SiHa cells.[12] Likewise, two independent studies reported anticancer properties of S. cumini seed-derived extracts in different cancer cell lines including ovarian cancer.[14],[28]

Among the three active phytochemicals identified in this study, the anticancer activity of OA is consistent with the previous report showing antiproliferative activity of OA extracted from different parts of Syzygium aromaticum in different cancer cell lines including SKOV-3 ovarian cancer cells.[29],[30] GA, a natural phenolic compound found in several medicinal plants, has been shown to possess anticancer activities.[31] However, S. cumini-derived GA has not been tested for its anticancer activity in any cancer cell line including ovarian cancer. QC, an important bioflavonoid present in more than 20 plants possessing antioxidant activity, is widely used in the treatment of metabolic and inflammatory disorders.[32],[33] The anticancer activity of QC has previously been documented;[33] however, like GA, the anticancer property of QC derived from S. cumini has not been reported earlier, especially in the ovarian cancer cells.


 > Conclusion Top


This study was undertaken to explore the anticancer properties of S. cumini-derived phytochemicals. Our data are consistent with the published reports, thus supporting the potential of developing these phytochemicals as anticancer therapeutic agents. The noncytotoxic effect of these phytochemicals in normal cells further supports this argument. The use of these phytochemicals in combination with the conventional chemotherapeutic agents is another potential area, which needs to be explored to develop better treatment regimens.

Acknowledgment

The authors are thankful to the Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Canal Road, Jammu, for their valuable input. AB is thankful to UGC for the financial support under the UGC-Startup program.

Financial support and sponsorship

UGC-Startup Grant to Dr. Audesh Bhat supported the study.

Conflicts of interest

There are no conflicts of interest.



 
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