GC-MS Evaluation, Antioxidant Content, and Cytotoxic Activity of Propolis Extract from Peninsular Malaysian Stingless Bees, Tetrigona Apicalis

Introduction Propolis has been used traditionally in several countries for treating various diseases as it possessed healing properties including antioxidant and anticancer qualities. In Peninsular Malaysia, Tetrigona apicalis is one of the species of stingless bees mainly found in virgin jungle reserves which largely contribute to propolis production. Therefore, this study is designed to evaluate the phytochemical contents, antioxidant properties, and the cytotoxic effect of ethanolic crude of propolis extract against MCF7 and MCF 10A cell lines. Method The ethanolic extract of propolis (EEP) was extracted using 80% ethanol. Identification of phytochemical contents and antioxidant properties of EEP was analysed by gas chromatography-mass spectrometry (GC-MS) and using 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method, respectively. The EEP cytotoxic activity was evaluated on MCF7 and MCF 10A using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Results Phytochemical contents of EEP demonstrated 28 compounds in which caryophyllene (99%), β-amyrin (96%), α-amyrin (93%), and caryophyllene oxide (93%) were the main compounds. The percentage of ABTS+ scavenging activity of EEP showed an inhibition of 9.5% with half-inhibitory concentration (IC50) value of 1.68 mg/mL. The EEP reduced MCF7 cells viability at IC50 value of 62.24 μg/mL, 44.15 μg/mL, and 32.70 μg/mL at 24, 48, and 72 hours, respectively. The IC50 value of MCF 10A was 49.55 μg/mL, 56.05 μg/mL, and 72.10 μg/mL at 24, 48, and 72 hours, respectively. The EEP cytotoxic effect of T. apicalis was more selective towards MCF7 at 72-hour incubation with a selectivity index (SI) of 2.20. Conclusion The EEP has been shown to have antioxidants and potential bioactive compounds and inhibited proliferation of the MCF7 cells. Further studies on the EEP role in the apoptosis pathway and its screening towards other cell lines will be evaluated.


Introduction
Propolis is popularly known as one of the traditional herbal medicines used worldwide. Propolis is composed of a collection of sticky resinous materials from different plant sources that function as a sealant for hole or cracks in the beehive. e word propolis comes from the Greek word "pro" meaning barrier or in defence and "polis" meaning city, or in full "defence of the city (or the hive)" [1]. Propolis has been effectively used in folk medicine since ancient times to treat cold sores and abscesses. Fortified with its unique pharmacological properties, several studies such as antiinflammatory, antioxidant, antifungal, antibacterial, antihepatotoxic, and anticancer have been done and successfully published [1][2][3].
Research on propolis has become the topic of interest due to its bioactive compounds and vital biological activities, particularly in Southeast Asian countries [4]. In Malaysia, multiple researches have been conducted to observe the effectiveness of propolis for some biological activities. e detection of phytochemical compounds and antioxidant activity of Malaysian propolis was successfully discussed in several studies using gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography (TLC) [5][6][7][8][9].
e abundance of stingless bee species in Malaysia, 17 to 32 of them well-known [10], has led to many opportunities to research each type of propolis, based on its precise species.
is study focuses on Tetrigona apicalis. Tetrigona apicalis first mentioned by Wallace and Smith [11] can be found mainly in the subtropical areas of Southeast Asia and Indo-Malaya/Australasia [12]. T. apicalis was selected for this study, as this particular species can easily be found in virgin jungle reserves, especially in Peninsular Malaysia [13]. Unlike other main species of Malaysian stingless bees that are normally kept for meliponiculture, such as Heterotrigona itama and Geniotrigona thoracica [10], T. apicalis is native to the wild and known for its efficacy as a potent pollinator group in most ecosystems [14].
To date, there exists no study on the cytotoxicity of T. apicalis on MCF 7 (breast cancer cells lines), a hormonedependent cell, and MCF 10A, nontumour human mammary epithelial cell lines. e in vitro cytotoxic assays for both cell types are crucial to determine whether the T. apicalis propolis extract is capable of working as a potential anticancer agent and in turn reducing toxicity towards noncancerous cells [17]. At the same time, studies specifically focused on the phytochemical screening of T. apicalis propolis extract for the identification of bioactive compounds for antioxidant and anticancer properties are lacking.
us, this study aims to determine the phytochemical compounds of T. apicalis propolis extract along with ABTS + radical scavenging activity and cytotoxic effect against MCF7 and MCF 10A.

Sample Collection and Identification of T. apicalis.
Propolis from the T. apicalis was collected at Tanjung Malim, Perak, Malaysia, by collecting the bee nest's inner part as described by Bonamigo et al. [18] with some modifications. e sample was kept in a plastic container and labelled. In order to identify the species of the stingless bees, the bee samples from the hive were taken as well. Alcohol swabs (soaked with 70% isopropyl alcohol) with scanty drops of 5% glacial acetic acid were prepared and placed in a killing jar prior to bee collection. e bee sample was put into the killing jar and the cover was closed tightly. e dead bees were put into the specimen container containing silica gel for further identification. Identification of the sample was completed by Centre for Insect Systematics (CIS), School of Environmental and Natural Resource Sciences, Faculty of Science and Technology of Universiti Kebangsaan Malaysia (UKM).

Preparation of Propolis Extract.
Raw propolis samples with dust and the dead bees were removed physically from the samples.
e preparations of the sample were in accordance with the method by Kothai and Jayanthi [19] with minor modifications. About 10 g powdered sample of T. apicalis propolis was extracted using 80% ethanol and stirred continuously at 400 rpm for 24 hours. e suspensions of the samples were subjected to centrifugation at 3000 rpm for 10 minutes. e extract was filtered using filter paper and concentrated using a rotary evaporator. e extracts were stored in a −20°C freezer. ereafter, the extract was freeze-dried and reduced to powder form.

Gas Chromatography-Mass Spectrometry (GC-MS).
Agilent Technologies 6890N Network GC system was used for GC-MS evaluation of phytochemical contents of the T. apicalis propolis extract. Approximately, 1 mg of the extract was dissolved in 1 mL methanol before filtering with a 0.45 μm Whatman nylon syringe filter. e extract was injected automatically in a splitless mode. e starter temperature was placed at 70°C and kept for 2 minutes. e temperature was further increased to 160°C (with a rate of 10°C/min) and maintained for 5 minutes. Eventually, the temperature was escalated to 270°C (with a rate of 20°C/min) and stabilised for 8 minutes. e compounds were selected based on the comparison from the National Institute of Standards and Technology (NIST) library. e compounds that showed 80% similarity with chemical compounds from NIST were selected for this study.

ABTS + Radical Scavenging Assay.
Determination of free radical scavenging activity in the ethanolic extract of propolis (EEP) was conducted by the method as described by Ismail et al. [6], Campos et al. [20], and Vongsak et al. [21] with minor modifications. Initially, 7 mM aqueous solution of ABTS + and 2.45 mM potassium persulfate in water was prepared and reacted. e mixture was kept in the dark at room temperature for 12 to 16 hours. ABTS + radical solution was diluted by reacting 1 mL ABTS + radical with 50 mL ethanol to achieve the absorbance of 0.70 (±0.02) at 734 nm using a spectrophotometer (Biomate spectrophotometer, ermo Fisher Scientific, USA). Samples at concentrations ranging from 0.01 to 0.313 mg/mL were used. About 125 μL of ABTS + radical was mixed with 1.25 μL of samples in a 96-well plate. e mixture was then incubated in the dark for 6 minutes at 37°C. All sample concentrations were tested in triplicates. e percentages of scavenging effects were measured by the equation as follows [22]: where A 1 is the absorbance of control and A 2 is the absorbance of samples. Each concentration was done in triplicate, and the mean half-maximal inhibitory concentration (IC 50 ) value was counted as mean ± standard deviation (SD). e positive control (Trolox) was treated under the same conditions as the samples.

Cytotoxic Assay
e MCF7 cell line was cultured in complete RPMI-1640 medium (included with penicillin-streptomycin and FBS); meanwhile, the MCF 10A cell line was cultured in complete DMEM (horse serum, hydrocortisone, EGF, insulin, and PBS). Both cells were incubated in a 5% CO 2 at 37°C for 48 to 72 hours until 80% confluency.

MTT Assay.
MTT assay was used to determine the cytotoxic effect of the extract using the method described by Aziz et al. [23]. Both cell lines were seeded at a density of 1 × 10 4 cells/mL in 96-well plates. Both cells were incubated for 24 hours to allow for cell attachment and were treated with 100 μL of extracts. e extracts were freshly prepared beforehand by diluting 100 mg of EEP in 1 mL of 100% DMSO. An amount of less than 0.5% of DMSO was used in this step to prevent any toxic effect on the cell for any insoluble extracts [24]. Extracts with eight different concentrations ranging from 0.975 to 125 μg/mL, positive and negative controls, were prepared with three replicates to ensure the validity of the results. Tamoxifen was used as a positive control, while negative control used media alone with 0.5% DMSO.
Cytotoxicity extracts were recorded for each time point (24,48, and 72 hours). After incubation, 10 μL of MTT solution was added to each well plate and was incubated for another 4 hours to produce formazan. Each well was added with 100 μL DMSO. e purple colour formed due to the dissolved formazan with DMSO corresponded to the number of viable cells [9]. e absorbance was measured at 570 nm with a spectrophotometer. e results were calculated as the mean values and SD in triplicate. e measurement of cell viability along with the IC 50 was calculated using the formula as stated as follows [25]: where SI > 2 was considered as high selectivity as suggested by Rashidi et al. [26].

Statistical
Analysis. e statistical analysis was done systematically in three replicates, and these data were represented as mean values along with SD. Microsoft Excel was used to plot graphs (cell viability (%) versus concentration) in order to conclude the IC 50 of the extract groups and positive control groups. A nonparametric (Kruskal Wallis) test was used to correlate the viability of the cell (%) between the treatments with the negative control. All variables were evaluated via SPSS and Microsoft Excel with p < 0.05 considered as significant.

T. apicalis Propolis Extract.
e percentage of yield of crude extract was measured with its physical appearance and recorded. e crude extract appeared whitish, and the samples were in powder form. e EEP sample from crude ethanolic extract yielded 57%.

Gas Chromatography-Mass Spectrometry.
e results of GC-MS analysis are summarised in Table 1.

ABTS + Radical
Scavenging Activity. Figure 1 shows the different concentrations (0.01, 0.02, 0.039, 0.078, 0.156, and 0.313 mg/mL) of ABTS + and control in the form of a linear regression graph. e IC 50 value was determined by using a linear regression equation [27]. IC 50 is described as the total antioxidant necessary to decrease the initial ABTS + radical by half [28]. e IC 50 of EEP and Trolox is further described in Table 2.
Based on Figure 1 and Table 2, the IC 50 of EEP was 1.68 mg/mL with the maximal ABTS + radical scavenging Evidence-Based Complementary and Alternative Medicine activity at 0.313 mg/mL and 9.5% inhibition corresponded to Trolox (49.8%).  Based on Figure 2
GC-MS analysis in this study showed that the EEP of T. apicalis contains compounds with antioxidant and anticancer properties. erefore, ABTS + radical scavenging assay was selected to analyse the antioxidant activity of EEP since ABTS + has a rapid kinetic reaction and intense response to antioxidants [42]. As suggested by Ibrahim et al. [5], the increment of antioxidant activity can be observed in the increase of radical scavenging activity that corresponded to a lesser value of IC 50 of the extract. is is in agreement with our present study, where the EEP of T. apicalis possess inhibitory activity against ABTS + radical with IC 50 of 1.68 mg/mL although positive control (Trolox) was better with IC 50 of 0.31 mg/mL. e graph in Figure 1 also showed that the radical scavenging activities were augmented, corresponding to the increase in the concentration of EEP. Previous studies by Rosli et al. [8] and Asem et al. [14] have also reported that T. apicalis propolis was very active in scavenging ABTS + and DPPH radicals. ese suggest that EEP has the potential to combat oxidative stress which is strongly attributed to the existence of crucial antioxidant compounds detected by the GC-MS analysis.
e previous cytotoxicity study on EEP of T. apicalis by Mat Nafi et al. [9] reported that EEP did not exhibit cytotoxic activity against different cancer cell lines. MDA-MB-231, SK-UT-1, and HeLa for 72 hours with IC 50 of HeLa were 68 μg/mL. Since EEP of T. apicalis consists of many bioactive compounds, a lower IC 50 was predicted [23]. At [39], oxygenated sesquiterpenes, (b) β-caryophyllene oxide [40], and triterpenoids, (c) α-amyrin and (d) β-amyrin [41]. Evidence-Based Complementary and Alternative Medicine 7 effectiveness of EEP as discussed earlier. erefore, these two key points are essential in determining the best potential of EEP to act as a cytotoxic agent towards MCF7.

Conclusion
EEP has been shown to have a high antioxidant level and potential bioactive compounds and can inhibit the proliferation of the MCF7 cells. e highest cytotoxic activities which corresponded to the lowest IC 50 of MCF7 was obtained at 72 hours of EEP treatment with the best SI at the same time point. Further studies are recommended to determine the role of EEP in the apoptosis pathway and the effect of this extract on other cell lines.

Data Availability
e analysed data used to support the findings of this study are included within the article.

Additional Points
Key Points. is journal article provides the effectiveness of Tetrigona apicalis propolis extract in promoting cytotoxic effect towards breast cancer cell line (MCF7) and nontumour human mammary epithelial cell lines (MCF 10A). e propolis extract of T. apicalis exhibited significant antioxidant properties and potential bioactive compounds and was capable of inhibiting the proliferation of the MCF7 cells.
e highest cytotoxic activity of MCF7 was acquired at 72 hours of EEP treatment with the best SI at the same time point.

Conflicts of Interest
e authors hereby declare that they have no conflicts of interest.