Evaluation of the antimicrobial and anticancer properties of the fruits of Synsepalum dulcificum (Sapotaceae)

Purpose: To evaluate the antimicrobial and anticancer activities of the fruit of Synsepalum dulcificum (Sapotaceae) against six bacterial strains and on two different colorectal cancer cell lines, respectively. Methods: The dried fruit of the plant was extracted in a Soxhlet apparatus successively with petroleum ether and ethanol, and concentrated in a rotary evaporator to obtain petroleum ether and ethanol extract, respectively. Phytochemical screening was done on the two extracts. The antimicrobial effects of the extracts against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogene, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus vulgaris were evaluated using agar well diffusion technique while the anticancer effect of the ethanol extract was assessed on HCT-116 and primary colon epithelial (PCE) cell lines by MTT assay. Results: The results indicate that the petroleum ether extract of Synsepalum dulcificum fruits exerted stronger antimicrobial activity than the ethanol extract. The ethanol extract also showed significant anticancer activity (p < 0.05). The calculated half-maximal concentration (IC50) of the extract on HCT116 cells at 24, 48, and 72 h are 14.99, 8.97, and 8.54 μg/mL, respectively, while the IC50 of the extract on PCE cell lines at 24, 48, and 72 h are 236.25, 206.09, and 196.72 μg/mL, respectively. The extract was more toxic to cancer cells than to normal cells. Conclusion: The results of this study lend some justification for the use of the fruits of Synsepalum dulcificum as an antibacterial and anticancer agent.


INTRODUCTION
Bacteria have been linked to cancer by the induction of chronic inflammation and production of carcinogenic metabolites [1]. Helicobacter pylori infection is an example of inflammatory mechanism of carcinogenesis [2]. Reports on human carcinogenesis by bacterial metabolites are not consistent.
According to WHO (2019) [3], Cancer is a large group of diseases characterized by the growth of abnormal and uncontrolled growth of cells. It is the second leading cause of death globally, accounting for 9.6 million deaths in 2018. In men, cancer of the lung, prostate, colorectal, stomach and liver are the most common. The most common type of cancer in women are those of the breast, colorectal, lung, cervix and thyroid. Besides, cancer is a growing public problem in the world.
The use of herbal remedies is now being widely embraced in many developed countries. It is the most popular and available in the primary healthcare system [4]. Synsepalum dulcificum, popularly known as miracle fruit, magic fruit, miraculous or flavor fruit, belongs to the family Sapotaceae. [5.] It is used in Africa to sweeten acidulated maize bread (kankies), palm wine and pito, a beer made from fermented grains [6]. Although, many scientific reports are available on the pharmacological importance of the leaves and the stem of the plant, information on the antimicrobial and anticancer activities of the fruit of Synsepalum dulcificums is scanty. This study aims to evaluate the antimicrobial and anticancer activities of the fruit of the plant.

EXPERIMENTAL Collection, authentication of plant material, and preparation of plant extracts
Synsepalum dulcificum was bought from Inanam, Kota Kinabalu, Sabah, Malaysia in October 2019. It was authenticated by Mr Aidil, a botanist at Forest Research Institute of Malaysia (FRIM), Kepong, Selangor Darul Ehsan, Malaysia, and given voucher number of "PID 381118-31". A specimen of the plant was deposited in the same Institute.
The fruits of the plant were air-dried under shade, seeds were separated and the pulp, dried, and powdered. The powdered material (100 g) was extracted in a Soxhlet apparatus successively for 10 h. with 200 mL of petroleum ether and ethanol, and concentrated in a rotary evaporator to obtain petroleum ether and ethanol extracts. The extracts were filtered and concentrated in a rotary evaporator. The concentrated extracts were dried in a desiccator containing calcium chloride. The dry extracts were kept in the dark below 4 °C in airtight containers.

Screening phytochemicals
Phytochemical screening was undertaken using standard methods [7].

Preparation of nutrient broth and Muller-Hinton agar medium
Nutrient powder (1.3 g) was weighed and mixed with 100 mL of distilled water in a beaker. For Muller Hinton agar medium, Muller-Hinton powder (19 g) was weighed, mixed with 500 mL of distilled water. Each was stirred continuously to form a homogenous mixture medium.
They were further heated to aid the dissolution process, transferred into test tubes, plugged with cotton, and were sterilized in an autoclave at 15 psi at temperature of 121 °C for 15 min. After sterilization, the test tubes were kept in slanting position and incubated at 37.5 °C to preserve sterility.

Preparation of microbial sub-culture
Fresh microorganism culture was transferred into nutrient medium to create sub-culture microorganism. The microorganism culture was inoculated into sterilized nutrient medium using sterilized inoculation loop. The inoculant was incubated for 24 h at 37 °C in the incubation oven.

Determination of minimum inhibitory concentration (MIC)
The method of Balouiri et al was followed [8]. Two-fold serial dilution was performed on the extracts (20, 10, 5, 2.5, 1.25, 0.63, 0.31 and 0.16 mg/mL diluted DMSO). The extracts with equal amounts of nutrient broth were transferred into test tubes. Each tube was inoculated with a microbial inoculum prepared in the same nutrient medium. The tubes were incubated at 37 for 24 h °C. Minimum inhibitory concentration (MIC) was determined based on turbidity with 0.5 McFarland standard as reference.

Agar well diffusion assay
Agar plate surface was inoculated by spreading a volume of the microbial inoculum over the entire agar surface. A hole with 8 mm of diameter was made using a sterile cork borer. A volume of 100 μL of extract of different concentrations (5, 10 and 20 mg/mL) was transferred using micropipette into the hole on the agar medium. The plate was incubated for 24 h at 37 °C. The diameter of the zone of inhibition was measured after the incubation period [8]. Tetracycline (50 μg) was used as positive control and dimethyl sulphoxide (DMSO) as negative control.

Plant extracts dilution
Ethanol extract of the fruit of Synsepalum dulcificum fruit (2 mg) was scraped out using a sterile spatula and weighed using an analytical mass balance before being put into a clean and sterile Eppendorf tube. The Eppendorf tube was then transferred into a laminar air flow and diluted with 1 mL DMSO to obtain a stock solution (2 mg/mL). The Eppendorf tube containing the extract was then sealed using aluminum foil. The stock solution was diluted with fresh media to obtain different concentrations (8.7, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL) of the extract.

Preparation of cell culture media
The medium used in cell culture was RPMI (Roswell Park Memorial Institute) 1640 with 10 % FBS and 1 % penicillin-streptomycin as antibiotic source. RPMI (Roswell Park Memorial Institute) 1640 medium (250 mL) was prepared. FBS (10 %, 25 mL) was pipetted using a sterile serological pipette carefully. Penicillinstreptomycin (1%, 2.5 mL) was pipetted and added into medium using serological pipette. The Schott bottle was gently stirred to mix he supplements well with the medium. The volume of media was topped up to 250 mL. The prepared medium was stored at 4 °C in a refrigerator.

Cell culture
Colorectal cells (HCT-116 and PCE cell) were obtained from the Department of Medical Microbiology, University of Malaya (UM), Malaysia. The medium used was RPMI 1640 with 10 % FBS and 1% Penicillin-streptomycin. The cells were cultured in T25 flask at 37 °C in a humidified atmosphere at 37 °C in which the CO2 level was maintained at 5% for 48 h.

Cell seeding
The confluency of cells was about 80 to 90 %. The medium was removed, and the cells were washed with PBS solution 3 times. The cells were then incubated with trypsin for 5 min to detach the cell from the surface and was centrifuged for 5 min. It was then removed from the tube and left with the pellet. The medium was added again into the falcon tube with pellets and mixed. Each exponential growth phase colorectal cancer cells was seeded, 1000 cells per well (HCT-116 and PCE), into 96-well plates and was incubated for 48 h. The number of viable cells per well was calculated using a haemocytometer.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay MTT (5 mg/mL) solution was added to the 96well plate and incubated at 37 °C for 4 h. The cell suspension was then removed from each well carefully using micropipette. Dimethyl sulphoxide (100 μL) was added into each well and was covered with aluminum while shaking by using microtiter plate shaker for 15 min.

Quantitation of MTT cell proliferation assay
The 96-well plate was loaded into Glomax microplate reader to determine and record the absorbance of the content of each well using a wavelength of 570 nm with reference wavelength of 630 nm. The percentage of cell viability was determined based on the dose response relationship which describes the interaction and change in effect on the cells. The results are calculated as the percentage of viability in relation to the untreated cells.

Statistical analysis
All tests were carried out in duplicates. Data are expressed as mean ± SEM, and were analyzed using Statistical Package for the Social Sciences (SPSS) version 22.0. One-way analysis of variance (ANOVA) and post hoc test with Turkey HSD were conducted.

RESULTS
The percentage extract yields for petroleum ether and ethanol were 6.30 and 13.86 %. The results of phytochemical screening are summarized as in Table 1. Table 2 shows the average zone of inhibition and standard deviation of different Synsepalum dulcificum extracts (petroleum ether and ethanol), tetracycline (0.05 mg/mL) and negative control (dimethyl sulfoxide) against different bacterial strains at different concentration.   The minimum inhibitory values for selected bacteria are shown in Table 3. Table 4 shows the results of the effect of the fruit of Synsepalum dulcificum on cell viability of HCT-116 cancer cells. The result of the effect of the fruit of Synsepalum dulcificum on cell viability of the primary colon epithelial (PCE) cells is shown in Table 5.

DISCUSSION
Results of the phytochemical screening showed that the ethanol extract of Synsepalum dulcificum contained more phytoconstituents than the petroleum ether fraction. A report of the quantitative phytochemical composition of S. dulcificum showed relatively high concentrations of flavonoids, tannins and saponins [9].
Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentration (MIC) will inhibit growth but not necessarily kill the microorganism.
The MIC values of the two different extracts of Synsepalum dulcificum fruits against the growth of both gram-positive and gram-negative bacteria ranged from 0.156 to 2.500 mg/mL. For petroleum ether extract, all bacteria strains except Pseudomonas aeruginosa displayed the same high sensitivity. The growth inhibition effects of the extract on the bacteria was observed at 0.156 mg/mL. The petroleum ether  Ethanol extract was used in the evaluation of anticancer activity on Synsepalum dulcificum fruits through MTT-assay against colorectal cell lines HCT-116 and primary colon epithelial (PCE) cells because of its ability to dissolve polar and non -polar substances [10]. The results showed that ethanol fruit extract of Synsepalum dulcificum exhibited potent anticancer activities on both the cell lines after the treatment period (24, 48 and 72 h). In the test against HCT -116 cell lines, the effect of the extract from the concentration of 15.63 to 1000 μg/mL was comparable with that of the positive control after 24, 48, and 72 h. While in the test against PCE cells, the result was comparable with that of the positive control at 24 and 48 h from 250 -1000 μg/mL.
In had been reported that the methanol and ethanol extracts of stem, and ethanol extract of the berry of S. dulcificum were cytotoxic to HCT-116 and HT-29 human colon cancer cells, but none of the extracts was cytotoxic to the THP-1 monocytic leukemia cells and HDFn normal human dermal fibroblasts. For both HCT-116 and HT-29, these extracts were significantly upregulated the expression of c-FOS and c-JUN, compared to the untreated negative control [11].
Phytochemicals such as polyphenols, flavonoids, alkaloids, and terpenoids are known to possess anticancer activity. The exact mechanism by which phytochemicals exert anticancer activity is still a topic of research. However, they are known to exert wide and complex range of actions on nuclear and cytosolic factors of a cancer cell [12,13].
The anticancer activity of the fruit of the plant may therefore be ascribed to the presence of lupeol, oleanolic acid, and ursolic acid in the fruit of the plant. Alpha-amyrin and β-amyrin may also contribute to its antimicrobial activity. However, further studies are required to confirm these.

CONCLUSION
The results indicate that petroleum ether extract of Synsepalum dulcificum fruits has superior antimicrobial activity to the ethanol extract. The ethanol extract also exhibits significant anticancer activity and may therefore be useful as an anticancer agent against colorectal cancer.