Abstract

In this research, we investigated the inhibitory effect of the ethanolic (EtOH) extract of bignay (Antidesma bunius) fruit on the viability of breast cancer cells (MDA-MB-231) by CCK-8 assay. The results showed that the half inhibitory concentration (IC50) was 219 μg/mL after the cells were treated with EtOH extract for 72 hours. The EtOH extract was sequentially extracted by petroleum ether (PET), ethyl acetate (EtOAc), and n-butanol (nBuOH). Among them, the EtOAc fraction showed higher anti-proliferative activity than the other fractions. Wound healing experiment showed the EtOAc fraction inhibited the migration of MDA-MB-231 cells and significantly delayed the transition of the G1 to S phase compared to control. UHPLC-MS/MS analysis showed that in addition to citric acid and phenolic acids (3-Hydroxybenzoic acid, aspirin, fertaric acid), EtOH extract of bignay fruit contains active ingredient like coumarin, genistin, amentoflavone, and luteolin 7-galactoside. The anti-proliferation activity of amentoflavone against MDA-MB-231 cells was also confirmed.

1. Introduction

Bignay (Antidesma bunius (L.) Sprengel) also known as Wu-Yuer-Cha in China and Mao-Luang in Thailand, belongs to Euphorbiaceae family, Phyllanthoideae subfamily ¹. There are about 170 plant species contained in the genus Antidesma and are distributed in tropical regions of the eastern hemisphere ². The plants of genus Antidesma are important ethno-medicinal plants in Thailand, the Philippines, and Indonesia ³. The entire plant of A. bunius is of medicinal value due to its antioxidant, anticancer and antidiabetic activities ⁴. There are 16 species of Antidesma genus plants in China, mainly distributed in Guangdong, Guangxi, Fujian, Guizhou, Yunnan and Hainan provinces. It often grows as a wild plant or is used as a green ornamental tree. The Chinese traditional medicine books "Color Atlas of Commonly Used Chinese Herbal Medicines" records that the roots, fruits, and leaves of A.bunius has the medicinal activities of convergence, quenching diarrhea, quenching thirst and promoting blood flow. Edible fruits of A. bunius are used to make juice or red wine rich in phytochemicals with enhanced health-promoting properties ⁵. Despite the presence of phenolic acids, flavonoids and anthocyanins in the fruit of A. bunius ⁶, its anticancer effect has not been fully elucidated.

Breast cancer is the kind of cancer with the highest incidence in women in the world today, and about 2 million new cases and 500,000 deaths in 2018 ⁷. The MDA-MB-231 cell line is a well-known malignant breast cancer model, due to its high invasiveness and motility and its ability to develop tumors and metastasize in in vivo assays in rodents ⁸. Currently, chemotherapy is the most effective treatment for cancer-suffering patients, and there is a constant need to find new therapeutic drugs and treatment methods to fight this disease. Paclitaxel, originally extracted from the bark of Taxus brevifolia, is a drug commonly used to treat breast and ovarian cancer, but it has the disadvantages of low water solubility and large adverse reactions ⁹.

Thus, research dedicated to finding novel compounds to inhibit any of the acquired features of cancer cells are of high value. In addition to this topic, our in vitro research indicates that the fruit extract of A. bunius prevents breast cancer cell proliferation and migration. This result provides an empirical basis for using fruits of A. bunius as food with anticancer effects.

2. Materials and Methods

Fruits of A. bunius were collected from Wanning, China and were certified in August 2019 by the botanist Rong-tao Li from the Institute of Medicinal Plants (Hainan Branch), Chinese Academy of Medical Sciences. The red colored fruit (2 kg) were dried in an oven at 40°C for 72 h. Later, it was grounded and extracted with 80% ethanol (EtOH) for 24 h. The sample was sonicated 3 times (30 min each time) in a sonicator by maintaining 25°C bath temperature. Extract was dried by rotary evaporation at 40°C and then by using a freeze dryer. Stock solution was prepared by dissolving 0.2 g of the dried extract in 2 mL DMSO, filtered with a filter (0.22 μm) and stored at -20°C until use.

In a second phase of extraction, the stock solution was dissolved in ultra-pure water, and sequentially extracted using petroleum ether (30-60°C boiling range, PET), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Each fraction was separately freeze-dried to obtain PET, EtOAc, n- BuOH and water extracts.

Human breast cancer cell line (MDA-MB-231) was received from Kunming Cell Bank, Chinese Academy of Sciences (Kunming, China) and was cultivated with RPMI 1640 media (Gibco, USA) containing 10% fetal bovine serum (BOSTER, Wuhan, China) and 1% streptomycin/penicillin (Biosharp, Hefei, China). 25 mM HEPES (Yuanye, Shanghai, China) was added to cell culture when performing experiment with the fruit extract. Cultures were maintained in humidified incubator (Thermo, USA) at 37 °C with 5% CO₂.

Cells were seeded at the rate of 2000 cells per well in 96-well microplate. After 24 h, media containing different concentrations of A. bunius fruit extract was added. As a control, media with 0.1% DMSO was used. After treatment, cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) (BOSTER, China) following the company’s recommended protocol. Briefly, media was gently removed after 72 h of treatment, cells were washed with phosphate-buffered saline (PBS) and then 90 μL of medium plus 10 μL of CCK-8 were added. The cell culture plate was put back in the incubator for 2 h. Finally, the absorbance was determined at 450 nm with a microplate reader (Thermo, USA). To calculate the relative cell viability, the following formula was used:

Extracting solution (methanol: water = 1: 1, with internal standard) was added in 2 mg of extract. LC-MS/MS analysis were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 µm) coupled to Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase was 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.7) (A) and acetonitrile (B). The analysis was carried with gradient elution as follows: 95% B for 0-0.5 min; 95-65% B for 0.5- 7.0 min; 65-40% B for 7.0-8.0 min; 40% B for 8.0-9.0 min; 40-95% for 9.0-9.1 min and 95% B for 9.1-12.0 min. The temperature of the column was 30°C. The temperature of the auto-sampler was 4°C and the volume of the injection was 2 μL.

The QE HFX mass spectrometer with acquisition program (Xcalibur, thermo), was used to acquire MS/MS spectra in the information-dependent acquisition (IDA) mode. The ESI source conditions were set as following: Aux gas flow rate as 25 Arb, sheath gas flow rate as 30 Arb, capillary temperature 350°C, MS/MS resolution as 7500, full MS resolution as 60000, spray Voltage as 3.6 kV (positive) or -3.2 kV (negative), collision energy as 10/30/60 in NCE mode.

Using Proteo Wizard, the raw data was converted to mzXML for processing with an in-house software developed using R and based on XCMS for peak detection, extraction, alignment and integration. Then an in-house MS2 database (Biotree DB) was applied in metabolite annotation. Subsequently, an in-house MS2 database (Biotree DB) was used for metabolite annotation. The annotation cutoff was fixed at 0.3.

In vitro cell migration can be calculated by a scratch assay that is appropriate, inexpensive and commonly used to mimic cell migration during in vivo wound healing ¹⁰. MDA-MB-231 cells (2.2×10⁵) were seeded per well in 24-well plates for 24 h before performing a wound with a 200 μL sterile tip. Cells were gently washed with PBS to remove floating cells, and media containing 2.5% fetal bovine serum (FBS) with and without fruit extract was added. Media was removed and cells were fixed after staining with 0.1% crystal violet (Yuanye, Shanghai, China). Pictures of the same field were taken at 0 and 24 h. Cell-free areas were determined using ImageJ software. The % relative migration was calculated with the following formula:

Each well of 6-well plate was seeded with 5×10⁵ MDA-MB-231 cells, and cultivated with 50 µg/mL EtOH extract. After 24 h of treatment, cells were detached with trypsin without EDTA, washed twice with pre-cooled PBS. Then, cells were fixed with 70% ethanol, and refrigerated overnight at -20°C. Later, cells were washed twice with pre-cooled PBS, and 2 μL ribonuclease (RNaseA) at a concentration of 1 mg/mL was added, and kept at 37°C for 30 min, then 400 μL propidiun iodide (PI) staining solution (KeyGen, JiangSu, China) was added and kept in the dark for 30 minutes. Flow cytometry (BD FACS Calibur, USA) detected amount PI per cell and the Modfit LT 5.0 software (Variety Software Home, USA) calculated the percentage of cells in each cell cycle phase.

The EtOAc extract and amentoflavone (Yuanye, Shanghai, China) standard were analyzed by Agilent 1200 HPLC (Agilent, USA) with an UV detection, SB-C18 column, using methanol (A) and 0.5% formic acid aqueous solution (B) as mobile phases, and gradient elution (0~20 min, 40~60% A; 20~40 min, 80% A; 40~60 min, 40% A). The flow rate and injection volume were 0.4 mL/min and 10 μL, respectively.

All experiments were repeated more than 3 times. One-way ANOVA and Dunnett’s multiple comparisons test were applied by using GraphPad Prism version 8.0.1.

3. Results

Altogether 14 g of extract powder were obtained from 0.29 kg dried fruits of A. bunius by ethanol extraction. EtOH extract was dissolved in DMSO, diluted to a certain concentration in DMEM medium, and tested for its proliferation inhibitory activity on breast cancer cell lines (MDA-MB-231). The results are shown in Figure 1. The anti-proliferative activity of EtOH extract showed a dose- dependent effect. The IC₅₀ of the EtOH extract of A. bunius fruits against MDA-MB-231 cells was determined to be 219 µg/mL after treatment for 72 h.

The 30 top content compounds are listed in Table 1. Most of these compounds were detected in the negative MS and only two compounds were detected in the positive MS (Figure 2). The compound in highest concentration was citric acid, which is the main acid in fruits like orange, passion fruit and pineapple. There are other phenolic acids like 3-hydroxybenzoic acid, 4- hydroxycinnamic acid, fertaric acid, aspirin, 2,3-dihydroxybutanedioic acid, itaconic acid, gallic acid, terephthalic acid and vanillic acid. Coumarin and flavonoids like genistin, amentoflavone and luteolin 7-galactoside are also in high content in EtOH extract and may play an important anticancer activity.

200 μg/mL of PET, EtOAc, n-BuOH and water extracts were tested to evaluate their inhibitory activity on the viability of MDA-MB-231 cells. Among these fractions, EtOAc extract exhibited the highest inhibition of cell viability and the aqueous phase showed the lowest activity (Figure 3).

In the wound healing process, cell migration and proliferation play a significant role. The effect of EtOAc extract on the migration of MDA-MB-231 cells was also examined. Migration of these cells was interrupted by increasing concentration of EtOAc extract in a dose dependent manner (Figure 4). Results showed that treatment with 100 μg/mL EtOAc extract strongly inhibited the motility of MDA-MB-231 cells towards the wound (Figure 4).

The 50 μg/mL EtOAc extract significantly increased the proportion of cells in G0/G1 phase after 24 h of treatment compared to control (Figure 5). The proportion of cells in G0/G1 phase was 58% in the control group while in the EtOAc treated extract reached to 63% (p<0.01). On the other hand, the cell proportion in S phase was 37% when treated with EtOAc compared to 32% in the control group (p<0.05). This result shows that the EtOAc extract phase of A. bunius fruit accumulates MDA-MB-231 cells in the G0/G1 phase.

From the HPLC analysis of the EtOAc extract fraction of A. bunius, the component that peaked at 49.2 min was isolated and identified as amentoflavone (C₃₀H₁₈O₁₀) by mass spectrometry. Molecular Weight of amentoflavone was 538.4579 (Figure 6).

Five different concentrations of amentoflavone (31.25, 62.5, 125, 250, 500 μg/mL) were tested to inhibit the viability of MDA-MB-231 cells. It was observed that the amentoflavone IC₅₀ against breast cancer cell’s viability was 192 μg/mL after treatment for 72 h. The results showed significant inhibition of the cancer cells at highest concentration i.e., 500 μg/mL. A slight non-significant increase on cell viability was observed with concentrations of amentoflavone lower than 100 μg/mL (Figure 7).

4. Discussion

Breast cancer accounts for 7%-10% of all malignant tumors and triple-negative breast cancer (TNBC) accounts for about 15-20% of breast cancer ¹¹. Intervention of traditional Chinese medicine at different stages of breast cancer treatment can effectively improve the cancer patients, life quality and reduce the rate of recurrence and metastasis ¹². Previous studies have shown that crude methanol extracts from leaves and fruits of A. bunius have cytotoxic activity against Artemia salina ⁵. Matured leaves of A. bunius have been used against snakebite and young leaves are boiled and used in syphilis and skin disorders in India ¹³. Ethanolic fruit extracts of A. bunius were used as herbal drug in diabetes therapy ¹⁴.

It was found that the polyphenolic compounds in fruit of A. bunius are mainly procyanidin B2, procyanidin B1, (+)-catechin, (–)-epicatechin, rutin and tran-resveratrol cyanidin-3-O-glucoside, and the contents of gallic acid, (-)-epicatechin, and (+)-catechin decreased during fruit ripening, and A. bunius fruit possess the highest antioxidants at over ripe stage ⁶. In this study, anthocyanins with wine red color mainly concentrate in water fraction, which showed no anti-proliferative activity to MDA-MB-231 cells. In our work, the high concentration of phenolic acid in our extract implies the red fruits we collected were not fully ripened, since the fully ripened fruit shows dark red color. The relationship between anti-cancer activity and the concentration of polyphenols needs to be further studied.

The G1 phase of the cell cycle involves mechanisms that allow gene expression for regular cell functioning and evaluation of DNA damage before the duplication of the genetic material in the S phase. The results of this study show that fruit extract of A. bunius delays MDA-MB-231 cells in the G1 phase that correlates with the decrease proportion of cells in the S phase. Altogether with the decrease in cell survival caused by exposure to A. bunius suggest that the extract inhibits cell proliferation by blocking or inhibiting cells in the transition from G1 to S phase. Probably, signaling pathways of cyclin D and cyclin E (regulators of entering the S phase) could be possible targets of A. bunius fruit extract.

MDA-MB-231 cells represent a great model of aggressive breast cancer since they have a high level of motility. Thus, finding compounds that preclude their cell motility are of great interest. Clinically, preventing cancer cell motility could retard cancer aggressiveness. As showed in this work, the extract of A. bunius fruit inhibits the migration of MDA-MB-231cells, pointing to a plausible use of this fruit extract as an anticancer agent.

Flavonoids are a large group of polyphenolic compounds present in a variety of fruits and vegetables with many biological properties. The flavonoids in the fruit extract of A. bunius such as coumarin, genistin, amentoflavone and luteolin 7-galactoside have already been proved to have multiple biological activities ^{15, 16, 17}. Amentoflavone is a naturally occurring biflavonoid compound abundant in Selaginella tamariscina, with anti-inflammatory ¹⁸, anti-oxidative ¹⁹, anti-microbial ²⁰, anti-apoptotic ²¹, anti-radiation ²², antitumor ¹², neuroprotective ^{23, 24} and cyano-bacterial killing effects ²⁵. Previously, the authors confirmed the anti- proliferative effect of the ethanolic extract of leaves of Antidesma montanum on the MDA-MB-231 cells and found that amentoflavone was present in this extract ²⁶.

The molecular mechanism underlying amentoflavone have been previously studied ²⁷. Amentoflavone inhibits TNF-α, VEGF, MMP-9, MMP-2, IL-6, and IL-1β expression and secretion by blocking NF-ĸB activation. ²⁸. The blockade of fatty acid synthesis triggered SK- BR-3 breast cancer cell apoptosis ²¹, and by regulating the Hedgehog/Gli1 signaling pathway in SUM159 stem cells of breast cancer and also inhibits tumor formation ²⁹. Amentoflavone has also been suggested as a potential adjuvant agent to boost the anti -cancer effect of doxorubicin ³⁰.

5. Conclusion

The ethanol extract of the fruit of bignay (A. bunius) possess anti-proliferative and anti- migratory effect on MDA-MB-231 breast cancer cells. There are abundant phenolic acids and flavonoids in the ethanol extract of bignay fruit. The main active substance concentrates in the ethyl acetate fraction, and it delays the G1 phase of MDA-MB-231 cells, probably causing apoptosis that could explain the reduced cell survival. Importantly, amentoflavone was identified as one active ingredient in fruit of A. bunius.

Acknowledgements

This study was financially sponsored by the Basal Research Fund of the Central Public-interest Scientific Institution of the Chinese Academy of Tropical Agricultural Sciences (grant number 1630092019002). We would like to thank Biotree Biotech Co., Ltd. (Shanghai, China) for providing technical assistance in performing the UHPLC-MS/MS analysis.

References