In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activities of Mesua beccariana (Baill.) Kosterm., Mesua ferrea Linn., and Mesua congestiflora Extracts

The in vitro cytotoxicity tests on the extracts of Mesua beccariana, M. ferrea, and M. congestiflora against Raji, SNU-1, HeLa, LS-174T, NCI-H23, SK-MEL-28, Hep-G2, IMR-32, and K562 were achieved using MTT assay. The methanol extracts of Mesua beccariana showed its potency towards the proliferation of B-lymphoma cell (Raji). In addition, only the nonpolar to semipolar extracts (hexane to ethyl acetate) of the three Mesua species indicated cytotoxic effects on the tested panel of human cancer cell lines. Antioxidant assays were evaluated using DPPH scavenging radical assay and Folin-Ciocalteu method. The methanol extracts of M. beccariana and M. ferrea showed high antioxidant activities with low EC50 values of 12.70 and 9.77 μg/mL, respectively, which are comparable to that of ascorbic acid (EC50 = 5.62 μg/mL). Antibacterial tests were carried out using four Gram positive and four Gram negative bacteria on Mesua beccariana extracts. All the extracts showed negative results in the inhibition of Gram negative bacteria. Nevertheless, methanol extracts showed some activities against Gram positive bacteria which are Bacillus cereus, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA), while the hexane extract also contributed some activities towards Bacillus cereus.


Introduction
Traditional medicines for human diseases have been widely used in many parts of the world. Herbal plants are usually the primary source of medicine in many developing countries. Natural product compounds from plants provide biologically active compounds, many of which have been developed as new lead chemicals for pharmaceuticals [1]. Only a limited number of plants have been scientifically explored from the many plant species worldwide. Cancer which is the uncontrolled growth of abnormal cells in the body [2] is a serious health problem. Treatments include surgery, chemotherapy, and radiation therapy. Many cancers have developed resistance to prolonged chemotherapy. Hence, there is a need to develop more effective and safer medicines such as herbal medicines. Free radical, a dangerous threat worldwide, causes aging and attacks and damages our immune systems which can lead to chronic inflammation of human cells and body. Recent reports revealed that free radicals provoke the neurodeterioration disorder of human beings which can lead to diseases such as Alzheimers [3] and Parkinsons [4,5] diseases. Therefore, antioxidants are required to reduce and prevent oxidative damage. Mesua species belong to the Clusiaceae family and consist of many health-promoting bioactive compounds [6][7][8][9][10]. Recent pharmacognosy analysis on Mesua ferrea leaf and fruit extracts showed both methanolic extracts to exhibit good antibacterial activity towards Staphylococcus aureus [11]. Previous research papers on Mesua species have shown that these plants exhibit many biological activities such as antioxidant [12][13][14], hepatoprotective [12], antiarthritic [15], immunomodulatory [16], antibacterial [17], antiacetylcholinesterase (AChE) [18], and larvicidal [19] activities, as well as antiproliferative effects [9,[20][21][22]. These plants were thus selected for our pharmacological investigation. The ultimate aim of this study was to discover plants with promising bioactivities which can then be developed into drugs through preclinical and clinical developments. Our ongoing research is focused on the screening of the extracts of Mesua beccariana, Mesua ferrea, and Mesua congestiflora against a panel of human cancer cell lines and DPPH scavenging agent. Furthermore, antimicrobial assay was also carried out using four Gram positive and four Gram negative bacteria on the Mesua beccariana extracts. Preliminary screening results of cytotoxic and antioxidant activities for Mesua beccariana, Mesua ferrea, and Mesua congestiflora will be reported. We have reported a novel cyclodione coumarin together with other known compounds from Mesua beccariana in a previous paper [23].

General.
All the apparatus used for cell culture were sterilized and decontaminated using Hirayama HICLAVE HVE-50. Cell culture handling was carried out in an ESCO Class II BSC Biosafety Cabinet. The healthy cells were spun down, adhered together, and separated from unhealthy and dead cells by using Thermo Scientific Sorvall ST 16R centrifuge machine. All cultures were incubated in 5% CO 2 humidified incubator at 37 ∘ C (ESCO Celculture CO 2 Incubator with model number CCL-170B-8). Cell stocks were placed in a −86 ∘ C ultralow temperature freezer (Scancool SCL 50 P) and preserved in a liquid nitrogen tank (Taylor-Wharton LS300).

Preparation of Plant Extraction.
The aerial parts of the plant samples were extracted using the conventional extraction method in which solvent was added and removed in batches. The dried and milled samples were soaked in nonpolar (hexane), medium polar (dichloromethane, or ethyl acetate) and polar (methanol) solvents in a polarity increasing order. The solvent was decanted and replaced with new solvent after 48 hours.
The air-dried and powdered Mesua beccariana stem bark, Mesua ferrea roots, and Mesua congestiflora stem bark were extracted successively with n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EA), and methanol (MeOH). The extracts were dried under reduced pressure using a rotary evaporator. Table 5 shows the weights of the extracts obtained from the three Mesua species.

Cytotoxicity Assay (MTT Assay).
Preliminary screening tests for cytotoxic activity against a panel of human cancer cell lines on the crude extracts and quercetin (standard) were undertaken by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetra zolium bromide) assay. The nine tested cancer cell lines were Raji (human B lymphocyte), SNU-1 (human gastric carcinoma), K562 (human erythroleukemia cells), LS-174T (human colorectal adenocarcinoma), HeLa (human cervical cells), SK-MEL-28 (human malignant melanoma cells), NCI-H23 (human lung adenocarcinoma), IMR-32 (human neuroblastoma), and Hep-G2 (human hepatocellular liver carcinoma). The Raji, SNU-1, K562, HeLa, Hep-G2, and NCI-H23 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), while the LS-174T, SK-MEL-28, and IMR-32 were maintained in MEM supplemented with 10% FBS. All the cell lines were cultured in 75 cm 2 T-flask and maintained at 37 ∘ C in 5% CO 2 humidified incubator. The MTT assay is based on the protocol illustrated by Mosmann [24] and was executed in 96-well flat bottom plates. The varying concentrations of 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 g/mL were prepared by a serial dilution method. An aliquot of 100 L of each substock with different concentrations was added to each well together with 100 L of selected cells to give concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, and 3.13 g/mL and made up to a final volume of 200 L in each well. 200 L of cells with no extracts (untreated cell control-positive control) and 200 L of medium only (blank medium-negative control) were prepared in the same plate. Each of the samples and controls were prepared in triplicates.
The plate was then incubated for 72 hours at 37 ∘ C in 5% CO 2 humidifier incubator. After 72 hours, 20 L of MTT solution was added to all the wells and incubated for 3 hours in a 5% CO 2 humidifier incubator. The plate was then spun at 3000 rpm for 10 minutes. 160 L of supernatant from each well was discarded and then added with 160 L of DMSO to dissolve the purple formazan crystals.
The absorbance of each well was determined using a microplate reader at 550 nm. The average absorbance of each crude extract was calculated, and the average value was used to determine the percentage of cell viability by using the following formula: Percentage of cell viability where A is the absorbance of sample, B is the absorbance of negative control, and C is the absorbance of positive control.
BioMed Research International 3 A graph of percentage of cell viability versus concentration was plotted for each extract. The half maximal inhibitory concentration (IC 50 ) values were obtained from the plotted graph. Further dilutions will only be performed on the extracts with IC 50 values less than 3.13 g/mL. Three independent experiments were conducted to assure the accuracy of the results. Quercetin was used as standard drug throughout the cytotoxicity experiments.

Antioxidant Assay
2.5.1. DPPH Scavenging Assay. The DPPH (2,2-diphenyl-1picrylhydrazyl) radical scavenging assay was used to evaluate the antioxidant property of the sample. The assay was performed in 96-well flat bottom plates. The DPPH solution was prepared in a stock solution of 5 mg/2 mL in ethanol (EtOH) and wrapped with aluminium foil. The varying concentrations of 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 g/mL were prepared by serial dilution method. An aliquot of 200 L of each substock with different concentrations was added to each well together with 20 L of DPPH solution. 200 L of EtOH and 20 L of DPPH solution (blank mediumnegative control) were prepared in the same plate. Each sample and the control were prepared in triplicates. The plate was then incubated in a dark room at room temperature for 30 minutes. The absorbance of each well was recorded after 30 minutes using a microplate reader at 517 nm. The average absorbance of each crude extract was calculated, and the average value was used to determine the percentage of total radical scavenging activity by using the following formula: Percentage of total radical scavenging activity where, is the absorbance of blank, and is the absorbance of sample. A graph of percentage of total radical scavenging activity versus concentration was plotted for each extracts. The half maximal effective concentration (EC 50 ) values were obtained from the plotted graphs. Three independent experiments were conducted to ensure the precision of the results. Ascorbic acid (vitamin C) was used as the standard drug.

Total Phenolic Content (TPC).
The TPC analysis is to analyze the total phenolic content present in the extract. The TPC analyses were carried out in 24-well flat bottom plates. The extract was initially oxidized by Folic-Ciocalteu reagent (FC) and subsequently neutralized by sodium bicarbonate, NaHCO 3 . Gallic acid was used as the standard in the experiment.
A standard curve was constructed using the equation = 0.0026 + 0.0402, where is absorbance and is gallic acid content in g/mL. This equation was used for each extract to determine their percentage of gallic acid content. The total phenolic contents of crude extracts were expressed as gallic acid equivalent ( g of gallic acid/mg of crude extracts).
An aliquot of 100 L of each solution with different concentrations of gallic acid was added into each well together with 750 L of FC reagent (previously diluted 10fold with distilled water). A blank control solution (EtOH without extracts) was prepared in the same plate. Consequently, 750 L of 60 g/L (60 g in 1 L distilled water) of NaHCO 3 was added into each well and left in the dark for 90 minutes. The absorbance of each extracts was measured at 725 nm. The experiment was executed in triplicate for accuracy. All the previous steps were repeated by different extracts with only one concentration which was 500 g/mL.
2.6. Antimicrobial Assay. Nutrient agar (NA) and Mueller Hinton agar (MHA) were prepared according to the manufacturer's instructions. The medium was cooled to 50 ∘ C after autoclaving, before approximately 25.0 to 30.0 mL of the medium was poured into 15 × 100 mm plastic petri dishes at a uniform depth of 4 mm. Each type of microorganism (from the stock culture) was streaked onto a noninhibitory NA plate to obtain isolated colonies. After an overnight incubation (16-20 hours) at 37 ∘ C, one single colony was selected and inoculated with a loop, transferred into 10.0 mL of sterile nutrient broth (NB), and incubated in a shaking incubator at 37 ∘ C for 16-20 hours. The density of bacteria was standardized using McFarland 0.5 turbidity standard to 1 × 10 8 coliform units (cfu)/mL by diluting each suspension in 10 mL of sterile distilled water to the appropriate density.
The antibacterial assay was performed using disc diffusion (Kirby-Bauer) method as described by Aksoy et al. The standardized bacterial suspensions were swabbed on MHA surface using sterile cotton wool. For initial screening, 1 mg of DCM : MeOH extract was loaded onto each Whatman No. 1 filter paper discs (ø, 6 mm) and were impregnated. The positive (streptomycin 10 g or vancomycin 30 g) and negative controls (blank disc impregnated with methanol) were used in the antibacterial test. The plates were left at 4 ∘ C for an hour to allow for the diffusion of the extracts before they were incubated for 16-20 hours at 37 ∘ C, and the diameter of the inhibition zones was then measured [25].

Results and Discussion
According to the National Cancer Institute (NCI), a crude extract can be considered active if it possesses an IC 50 value of less than 20 g/mL [20,26,27]. From this screening, Raji was found to have the lowest susceptibility cancer cells among the investigated cell lines. However On the other hand, the hexane and dichloromethane extracts of M. ferrea displayed moderate to strong antiproliferative effects against all the cancer cell lines except for   the Raji cells. The IC 50 values were in the range of 8.85 to 43.75 g/mL. However, the DCM extract barely gave any activity towards the HeLa cells, while the ethyl acetate and methanol extract, were inactive towards the proliferation of the tested cell lines.
Additionally, the hexane extract of Mesua congestiflora indicated mild cytotoxicities towards SNU-1, K562, and NCI-H23 cells and weak cytotoxic activity towards the rest of the cell lines. Both the ethyl acetate and methanol extracts were not cytotoxic towards all the investigated cells.
In conclusion, only nonpolar to semipolar extracts contribute to the cytotoxic effects on the panel of human cancer cell lines except for the polar extract of Mesua beccariana as shown in Table 1 (Figure 1). Quercetin, which was used as a standard, showed cytotoxic activity with IC 50 values ranging from 2.00 to 31.25 g/mL. In our previous paper, we reported eleven xanthones from the three plant species, and three of which were very cytotoxic towards all the nine cancer cell lines tested. The structure-activity relationship (SAR) study revealed that the diprenyl, dipyrano, and prenylated pyrano substituent groups of the xanthone derivatives contributed towards the cytotoxicities [22]. Moreover, preliminary tests of cytotoxicity on other isolated metabolites were also reported [9,21].
All the extracts of Mesua beccariana, Mesua ferrea, and Mesua congestiflora along with ascorbic acid (standard drug) at different concentrations were evaluated for their scavenging potentials on the DPPH free radical.
Several semipolar to polar extracts possess medium to strong scavenging activity against DPPH radical where the methanol (MeOH) extracts of Mesua beccariana and Mesua ferrea exhibited significant scavenging activity with EC 50 values of 12.70 and 9.77 g/mL which are comparable with ascorbic acid (EC 50 = 5.62 g/mL). The ethyl acetate (EA) extract of Mesua beccariana and Mesua congestiflora as well as the dichloromethane (DCM) extract of  Mesua ferrea reflected mild scavenging capability, while the dichloromethane extract of Mesua beccariana gave weak scavenging effect. Besides this, the hexane extract of all three Mesua species, the EA extract of Mesua ferrea, and MeOH extract of Mesua congestiflora were seen to be inactive towards DPPH scavenging agent. The EC 50 values for the extracts of three Mesua species towards DPPH free radicals are summarized in Table 2 ( Figure 2).
The total phenolic content (TPC) analysis was carried out on all the crude extracts by using Folin-Ciocalteu method.
The TPC values give information on the phenolic contents of the crude extracts and their antioxidant activities. The methanolic extracts of Mesua beccariana and Mesua ferrea exhibited the highest phenolic contents among the extracts with values of 363.82 and 441.33 g in gallic acid equivalent (GAE), whereas the ethyl acetate extracts of both species also  not tested not tested not tested 21.00 * Each data represents the mean of three independent experiments. Symbol "-" represents no activity. * * Each value represents the inhibition zone (mm). * * * 1-Bacillus cereus; 2-Micrococcus luteus; 3-methicillin-sensitive Staphylococcus aureus (MSSA); 4-methicillin-resistant Staphylococcus aureus (MRSA).  Table 3  Gram negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes were tested in this experiment. All the crude extracts showed negative results in the inhibition of Gram negative bacteria. The methanol extracts, however, showed some activity against B. cereus, MSSA, and MRSA. In addition, the hexane extract also gave weak activity against B. cereus bacterium. Table 4 summarizes the antibacterial activity of the crude extracts of Mesua beccariana.

Conclusion
Both DPPH radical assay and total phenolic content indicate that semipolar to polar extracts had high antioxidant properties, while non-polar extracts exhibited good cytotoxic activity. The pharmacognosy investigation showed adverse effects of crude extracts suggesting that the three Mesua species could be a good source for the development of lead compounds.