INTRODUCTION:

Breast cancer is the most common cause of death in women worldwide. Several factors that can increase the risk of breast cancer are age, menopause at an advanced age, first gestational age of 30 years or more, obesity, family history, history of benign tumors, and genetic factors.¹ Mutations of BRCA1 and BRCA2 are genetic factors that can cause abnormal cell growth and lead to cancer.²

Other genetic factors that play a role are EGFR overexpression which causes tumor enlargement and poor prognosis, and Bcl-2 overexpression that causes a decrease in cancer cell apoptosis.^3,4 The most common breast cancer treatment is chemotherapy, but its use induces uncomfortable side effects such as nausea, vomiting, peripheral neuropathy, myelosuppression, and alopecia.⁵ Therefore, plants can be used as supportive therapy for breast cancer. Syzygium cumini is known as “juwet” in Indonesia. Previous studies showed that Syzygium cumini have antibacterial, antidiabetic, antiurilithiasis, and analgesic activities.^6–9

Syzygium cumini seed extract had cytotoxic properties against the MCF-7 cell line with an IC₅₀ 110μg/ml.¹⁰ Another study showed that the methanol extract of Syzygium cumini fruit had cytotoxic properties against the MCF-7 cell line with growth inhibition of 70.12%.¹¹ Some secondary metabolites are exhibited antioxidant activity and have contributed to the cytotoxic effect of plant extract.^12,13 Previous studies have shown that Syzygium cumini has higher total phenolic content and better antioxidant activity (based on IC₅₀ DPPH assay) than grapes, which have been recognized as antioxidant-rich fruits by the public.¹² The purpose of the presents study is to investigate the total phenolic content and cytotoxic activity against 4T1 cell lines from seed, leaves, and pulp of Syzygium cumini.

MATERIALS AND METHODS:

The leaves, pulp, and seeds of Syzygium cumini were obtained from the Karang Pilang area, Surabaya, East Java, Indonesia, and determined at Balai Materia Medica, Batu, East Java. Breast cancer 4T1 cells were obtained from the Laboratory of Cancer Chemoprevention Research Center (CCRC) Gajah Mada University, Indonesia.

The culture media used were DMEM High Glucose (Gibco®), WFI (WIDA WITM Unicap), HEPES (Gibco®), NaHCO3 (Gibco®), HCl pro analysis (pa) (Gibco®), NaOH pa (Gibco®), micropipette tip, penicillin-streptomycin (Gibco®), fetal bovine serum (FBS) (Sigma®), phosphate-buffered saline (PBS) (Gibco®), trypsin-EDTA (Gibco®), and dimethyl sulfoxide (DMSO) pa (EMSURE®), MTT powder (Gibco®).

The materials used for extraction, phytochemical screening and total phenolic content determination are ethanol (EMSURE®), n-hexane (EMSURE®), ethyl acetate (EMSURE®), chloroform EMSURE®), acetone (EMSURE®), glacial acetic acid (EMSURE®), toluene (EMSURE®), aquades, n-butanol, AlCl₃, sulfuric acid (EMSURE®), gallic acid (Sigma®), Folin-Ciocalteu (Sigma®), sodium hydroxide (NaOH) (Sigma®), formic acid, and KOH.

The absorbance of total phenolic content was measured using UV-Vis spectrophotometer (Shimadzu UV-1800). Optical density of MTT assay was determined using ELISA reader (SPECTROstar).

Extraction:

A dried sample of each part was ground and soaked in 96% ethanol for 72 hours to obtain crude extract. Solvents were removed in a rotary evaporator at a temperature of 40⁰ Celcius.

Phytochemical screening:

Thin-Layer Chromatography was performed to identify secondary metabolites of Syzygium cumini using silica GF_254nm as a stationary phase and organic solvents as the mobile phase (Table 1). Some reagents were used to detect secondary metabolites in each sample. The TLC results were observed under UV lamp with wavelengths of 254nm and 365nm, then the Rf value was calculated.

Table 1: Mobile phase and reagents for phytochemical screening

Secondary metabolites	Mobile phase (%v/v)		Reagent
Secondary metabolites	Pulp and seed	Leaves	Reagent
Alkaloid	Ethyl acetate: aceton: fornic acid (4: 5,75: 0,25)	Toluene: ethyl acetate: diethylamine (7:2:1)	Dragendorff
Terpenoid		Chloroform: Acetone (8:2)	Anisaldehide- H₂SO₄
Flavonoid		n-butanol: glacial acetic acid: aquadest (8:2:10)	AlCl₃10%
Antraquinone		Ethyl acetate: ethanol: aquadest (100:13,5:10)	KOH 10%
Tannin	Gellatin test

Total phenolic content:

Measurement of total phenolic content was carried out referring to the study of Guediri et al¹⁴ with a slight modification. Gallic acid was used as a standard to determine total phenolic content. Ten milligrams of gallic acid were dissolved in methanol and diluted into several serial concentrations: 50, 60, 70, 80, and 90ppm. Each sample weighed 10mg and dissolved in methanol. Folin-Ciocalteau 7.5% was added to each solution and incubated for 8 min under light-tight conditions. Four milliliters of NaOH 1% solution were added and incubated for 1 hour. The absorbance of each solution was measured in a UV spectrophotometer at a wavelength 747nm. The calibration curve was derived from the result between the concentration of the gallic acid solution and its absorbance (Figure 1). Total phenolic content was calculated using a gallic acid calibration curve and expressed as gallic acid equivalent (GAE) in mg per gram sample.

Cytotoxic activity:

Cytotoxic effect of seed, leaves, and pulp of Syzgium cumini were performed using MTT assay. Treatment was divided into four groups: cell control, media control, solvent control, and extract group. Each extract was dissolved in aquadest and diluted into five serial concentrations: 400, 500, 600, 700, 800, and 900ppm.

Five thousand breast cancer 4T1 cell lines were transferred into 96 well-plates and incubated for 24 hours until the cell was attached. The attached cells were washed with 100μL of PBS, then 100μL of the extract solution was added. After 24 hours of incubation, each well was washed with PBS, and 5mg/ml of MTT solution was administrated. Incubation was performed for 4 hours; then, the reaction was stopped by adding 10 μL of DMSO. The absorbance of each well was measured using an ELISA reader at a wavelength of 595 nm. The formula calculates the percentage of cell viability:

RESULTS:

Phytochemical screening (Table 2) showed that ethanolic extract of Syzygium leaves contains alkaloids, terpenoids, flavonoids, anthraquinones, and tannins; ethanol extract of Syzygium cumini pulp contains alkaloids, terpenoids, and anthocyanin; and Syzygium cumini seed extract contains flavonoids, anthraquinones, terpenoids, and tannins.

Table 2: Phytochemical screening of leaves, pulp and seeds extract from Syzygium cumini

	Leaves	Pulp	Seeds
Alkaloid	+	+	-
Alkaloid	Rf 0.36; 0.5	Rf 0.06
Terpenoid	+	+	+
Terpenoid	Rf 0.53; 0.73	Rf 0.71; 0.67	Rf 0.72
Flavonoid	+	+	+
Flavonoid	Rf 0.66; 0.86	Rf 0.21 (anthocyanin)	Rf 0.29
Anthraquinone	+	-	+
Anthraquinone	Rf 0.76; 0.5		Rf 0.29; 0.67
Tanin	+	-	+

The calibration curve of gallic acid solution shows a linear correlation between concentration and absorbance value (Figure 1). The results showed that the total phenolic content in the seed was the largest, followed by leaves and pulp (Table 3).

Figure 1: Calibration curve of Gallic acid vs absorbance

Table 3: Total phenolic content of Syzygium cumini extract

Extracts	Total phenolic content (mg GAE/g)
Leaves	329.60 ± 20.37
Pulps	24.09 ± 0.57
Seeds	503.01 ± 9.21

Based on the results of the cytotoxic test using MTT assay on the 4T1 cell line, IC₅₀ value of Syzygium cumini seed, leaves, and pulps was 613.92±40, 48; 660.18±15.02; 732.68 ± 69.41µg/ml. According to One way ANOVA analysis, the cytotoxic activity of Syzygium cumini leaves, pulps, and seeds is not significant (p >0.05). The viability percentage of MTT assay from Syzygium cumini leaf, and pulps extract followed a dose-dependent manner (Figure 2).

Figure 2. Viability percentage of each extract concentration (▲: seeds; ■: pulps; ♦: leaves)

DISCUSSION:

Phytochemical screening showed that seed extract contains terpenoid, flavonoid, tannin, and anthraquinone (Table 2). The results of previous studies showed that seed extract also contained alkaloids.^15,16 In this study, the cytotoxic activity of the seed extract was more potent than the leaf and pulp extracts. The seed extract also had a higher total phenolic content than the leaves and pulp (Table 3).

The leaf extract contained alkaloids, terpenoids, flavonoids, anthraquinones, and tannins, with a total phenolic content 329.60 ±20.37 mg GAE/g and IC₅₀ 660.18±15.02 µg/ml. Previous research exhibited that the methanol extract of Syzygium cumini leaves contained total phenolic content 8.52 ± 0.055 mg GAE/g.¹⁷ While pulp extract contained alkaloids, terpenoids, and anthocyanin, with total phenolic content 24.09 ± 0.57 mg GAE/g and IC₅₀ 732.68 ± 69.41 µg/ml. From the results of this study, the cytotoxic activity of the extract of Syzygium cumini seeds, leaves, and pulp was proportional to the total phenolic content. Although the total phenolic content of pulp extract is the lowest, there are anthocyanin compounds that contribute to the cytotoxic activity. The results of previous studies also showed that the total phenolic content in the fruit extract was lower than leaves extract.¹⁸

The previous study showed that Syzygium cumini seed extract had cytotoxic properties against the MCF-7 breast cancer cell line with an IC₅₀ 110 µg/ml, and Syzygium cumini fruit extract had cytotoxic properties against the MCF-7aro cell line with an IC₅₀ 27 µg /ml.¹⁰ The 4T1 breast cancer cell line is more aggressive and has a higher metastatic potential than MCF-7 and MCF-7aro, so the IC₅₀ values of Syzygium cumini leaf, pulp, and seed extracts in this study were higher than in previous studies.¹⁹

Phenolic compounds inhibit the growth of breast cancer cells through several mechanisms: 1) increasing apoptotic activity; 2) decreasing cell proliferation; 3) angiogenesis; 4)anti-inflammatory. Syzygium cumini contains a resveratrol compound that can reduce the number of terminal end buds in DMDA-induced experimental animals. Resveratrol can also inhibit the activity of 5-LOX and COX-2.^20–22 Syzygium cumini also contains the flavonoid compound myristine.¹⁴ A previous study showed that Syzygium cumini pulp extract was able to inhibit free radical activity by 38.8% at a concentration of 1000 g/ml.²³

In cultured of noncancerous breast human cell line (MCF-10F), anthocyanins were able to inhibit the formation of BPDE-DNA adducts.²⁴ The administration of anthocyanins in MCF7 cell culture could inhibit cell growth, increase apoptosis and decrease the number of cells in the G2/M phase.²⁵ In addition, anthocyanins were also able to reduce the process of metastases and the regulatory activity of matrix metalloprotease in MDA-MB-231 cell culture.²⁶

CONCLUSION:

Syzygium cumini seed extract has the highest total phenolic content and potential cytotoxic activity. In fruit extracts, although the total phenolic content was lowest, the presence of anthocyanin compounds could contribute to the cytotoxic activity. Based on the results of this study, Syzygium cumini fruit extract has the potential as a dietary food to prevent cancer.

ACKNOWLEDGEMENT:

This research was funded by Institute for Research and Community Service, Universitas Brawijaya with contract number: 696.37/UN10.C10/PN/2020.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

ABBREVIATIONS:

UV-VIS: Ultravioler-visible; BRCA1: Breast Cancer gene; EGFR: Epidermal growth factor receptor; WFI: Water for injection; DMEM: Dulbecco's modified eagle medium; GAE: Gallic acid equivalent; OD: Optical density; IC: Inhibition concentration

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Received on 19.01.2022 Modified on 21.04.2022

Res. J. Pharmacognosy and Phytochem. 2022; 14(3):145-149.

DOI: 10.52711/0975-4385.2022.00027