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Abstract Aim: This in vitro study aimed to investigate the effect of propolis extracts from two different solvents on human submandibular salivary gland (HSG) tumor cell line. Materials and Methods: Propolis was extracted by dichloromethane (DCM) and hexane (HEX). Crude extracts were prepared from 6.25 to 200 µg/mL in Dulbecco’s modified eagle medium without serum. Flavonoid and total phenolic contents of crude extracts were measured using a modified colorimetric method. The cytotoxicity was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay. The statistics were analyzed by independent sample t-test. Results: Propolis extracts obtained using DCM and HEX exhibited comparable % yield (38.58 and 38.25) and physical characteristics and different amounts of flavonoid (0.439 ± 0.02 and 0.250 ± 0.01 mg catechin/g sample) and total phenolic compounds (3.759 ± 0.03 and 1.618 ± 0.03 mg gallic acid equivalents/g sample). The DCM group at 25, 50, 100, and 200 µg/mL as well as the HEX group at 50, 100, and 200 µg/mL significantly displayed a decrease in % cell viability and an increase in % cytotoxicity, compared with the untreated control group (P < 0.05). The DCM group showed the half-maximal inhibitory concentration (IC50) of MTT (42.93 ± 2.70) and LDH (34.94 ± 0.22). The HEX group showed the IC50 of MTT (61.30 ± 5.39) and LDH (42.32 ± 1.00). Propolis extracts obtained using both DCM and HEX are effective to inhibit HSG viability. Conclusion: Regarding to the cell morphological observation, MTT and LDH assays, propolis extracts obtained using DCM and HEX exhibited the cytotoxic effect on HSG tumor cell line. Based on our knowledge, this research demonstrates the first preliminary result suggesting propolis as a natural product of choice for salivary gland cancer prevention and therapy.
Keywords: Cytotoxicity, dichloromethane, hexane, propolis, salivary gland tumor
Introduction 
Head and neck cancer is one of the highly prevalent cancers worldwide and potentially increases due to changes in demographics and lifestyles. Various studies have demonstrated that salivary gland cancers are rare, constituting 3%–11% of all head and neck neoplasms and 0.2% of all malignancies. Nevertheless, it can cause patients’ low quality of life even after the complete salivary gland cancer treatment, resulting from the complications of salivary gland dysfunction.[1] Until now, the medical research has still been challenged to figure out an ideal cancer treatment. Using the natural product as a choice for cancer prevention and hopefully for a combined option with the conventional treatment is promising.
Propolis is a mixture of lipophilic materials collected from many plants and added to beeswax. Propolis from different sources demonstrates their main chemical components, which are flavonoids and phenolic acids.[2],[3] This compound has been reported for several biological activities such as antibacterial and antimycotic, wound-healing, anti-inflammatory, and antioxidant properties.[4]
Propolis has also shown the anticancer property in cell culture and animal models.[5],[6] In addition, propolis exhibited the different effect in several cancer cell types because of a variety of solvents.[7] The hexane (HEX) extract inhibited the proliferation of intestinal, breast, liver, lung, and gastric cancers.[8] The water extract decreased the cell proliferation and induced the necrosis of colon cancer.[9] The methanol extract diminished the growth of human pancreatic cancer cells.[10] The ethanol extract inhibited the cell proliferation of prostate cancer cells, colon cancer, and malignant melanoma cells.[11] Recently, the comparison of the effects of chloroform, HEX, and ethanol extracts on human colorectal cancer has shown that all extracts were able to inhibit cancer cells; however, the chloroform extract exhibited the most superior result.[12] Surprisingly, there is few research investigating the effect of dichloromethane (DCM)- and HEX-extracted propolis on oral cancer cells. For example, the study by Utispan et al. has reported that DCM-extracted propolis displayed the cytotoxicity against primary and metastatic head and neck cancer cell lines.[13] Recently, Wezgowiec et al. have demonstrated the anticancer effect of Polish extract by ethanol and HEX-ethanol on human tongue cancer cells.[14]
Although the anticancer property of propolis has been reported in several cancer cells, there is still a lack of knowledge about the propolis on salivary gland cancer, particularly mucoepidermoid carcinoma and adenoid cystic carcinoma, which are two types of common salivary gland cancers. Moreover, the current treatments consisting of surgery, radiotherapy, chemotherapy, or the combination still have a limited efficiency and can cause severe complication. Therefore, this study aimed to investigate the effect of propolis extracted by DCM and HEX on human salivary gland tumor cell line.The result from this study would preliminarily broaden a future opportunity to develop propolis as a natural product of choice for salivary gland cancer therapy.
Materials and Methods Propolis sample preparation
Propolis was collected from honeybees (Apis mellifera in the Dimocarpus longan garden, Lamphun, the northern Thailand). It was crudely extracted by two solvents separately, DCM and HEX in 1:5 ratio (w/v). The solution was filtrated in a shaker incubator (Stuart, UK) with a speed of 100 rpm in the dark at the room temperature for 24 h. The crude extracts were filtered and evaporated in a rotary evaporator. The extracts were lyophilized and kept in −20°C until use. The % yield was calculated as follows: (Actual weight of extract/total weight of propolis) ×100.
The stock solution (0.05 g/mL) was prepared in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) and filtered through sterile syringe filter with a 0.2 µm diameter (Whatman, UK). Different concentrations of propolis solution were prepared by the two-fold dilution for 200, 100, 50, 25, 12.5, and 6.25 µg/mL in Dulbecco’s modified eagle medium (DMEM) (Gibco, USA) without serum mixed with 0.4% (v/v) DMSO.
Estimation of flavonoid content
The flavonoid content of the sample was determined according to the method of Wolfe et al.[15] A volume of 0.25 mL of a known dilution of test sample was mixed with 1.25 mL of reverse osmosis (RO) water and 0.075 mL of 5% sodium nitrite solution and incubated at room temperature for 5 min. Then, 0.15 mL of 10% aluminum chloride was added. After 6 min, 0.5 mL of 1M sodium hydroxide was added, and the mixture was diluted with another 0.275 mL of RO water. The absorbance of the reaction mixture was measured at 510 nm with a Shimadzu spectrophotometer and compared with a standard curve of prepared catechin solutions in ethanol. Using the standard curve, the total flavonoid content was expressed as milligrams of catechin equivalents per 1 g sample (mg catechin/g sample).
Estimation of total phenolic content
The total phenolic contents of the sample were measured using a modified colorimetric Folin-Ciocalteu reagent according to the method of Wolfe et al.[15] A volume of 125 µL of a known dilution of test sample was mixed with 500 µL of RO water and 125 µL of Folin-Ciocalteu reagent and incubated at room temperature for 6 min. Then, 1250 µL of 7% sodium carbonate and 1000 µL of RO water were added, and then the mixture was allowed to stand for 90 min. The absorbance of the reaction mixture was measured at 760 nm with a Shimadzu spectrophotometer and compared with a standard curve of prepared gallic acid solutions in ethanol. Using the standard curve, the total phenolic content was expressed as micrograms of gallic acid equivalents per 1 g sample (mg GAE/g sample).
Cell culture
Human submandibular salivary gland (HSG) cell line was cultured at 37°C, 5% CO2 in growth medium containing DMEM with 4.5 g/L glucose, L-glutamine, and sodium pyruvate, 10% fetal bovine serum, 100 units/mL penicillin with 100 μg/mL streptomycin (Gibco, USA). In a 96-well culture plate, 10,000 cells/cm2 were plated (Thermo scientific, China) in a growth medium at 37°C, 5% CO2 for 24 h. The next day, cultured cells were treated in different concentrations of propolis extracted by various solvents for 24 h. The experimental groups were propolis-treated cells, whereas the control group was untreated cells. The photographs of untreated and treated cells were taken before further analysis.
MTT assay
3- [4, 5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium (MTT) assay was performed with cells in a 96-well plate. Cells were washed with 100 µL of phosphate buffer saline (PBS) after the media removal. Cells were added to 200 µL of 0.05% MTT (Invitrogen, USA) in serum-free DMEM at 37°C, 5% CO2 for 2 h in dark. Then, cells were washed twice with 100 μL of PBS and the formazan crystals in cells were dissolved by 100 μL of DMSO. Next, the optical density (OD) at 540 nm was read by the microplate reader (BioTek, USA). The % cell viability was calculated as follows: [(OD treated−OD blank)/(OD untreated−OD blank)] × 100.
LDH assay
Lactate dehydrogenase (LDH) assay was conducted with cells in a 96-well plate by CytoTox 96 nonradioactive cytotoxicity assay (Promega, USA). The supernatant of cells was collected. From each sample, 50 µL of supernatant was mixed with 50 µL of assay buffer and incubated at the room temperature for 30 min in dark. Then, the reaction was stopped by adding 50 µL of stop solution before reading the OD at 490 nm. The % cytotoxicity was calculated as follows: (OD cell of LDH release−OD no cell of LDH release/OD maximum LDH release−OD no cell of LDH release) × 100.
Statistical analysis
The half-maximal inhibitory concentration (IC50) was calculated by using CurveExpert 1.4. The statistic was analyzed by SPSS version 18 with independent sample t-test to compare the differences between groups (propolis extract group versus untreated control group) at the 95% confidential interval.
Results Propolis that was extracted by various solvents demonstrated the different % yield of dry weights and physical appearances [Figure 1]. The percent yields of DCM and HEX were 38.58% and 38.25%, respectively. The crude extracts from both solvents also exhibited sticky materials in yellow color. Propolis extracts obtained using DCM and HEX showed the flavonoid compound by 0.439 ± 0.02 and 0.250 ± 0.01 mg catechin/g sample, as well as the total phenolic compound by 3.759 ± 0.03 and 1.618 ± 0.03 mg GAE/g sample, respectively [Table 1].
Figure 1: The % yield and physical characteristics of propolis obtained using from dichloromethane and hexane. The % yield is the ratio of the actual dry weight of the extract to the total weight of propolis, expressed as a percentage
Table 1: Flavonoid and total phenolic compound contents of propolis extracts from two different solventsUntreated cells (0 µg/mL or control group) were polyhedral, indicating the regular epithelial cell morphology [Figure 2]A. Treated cells with 6.25 and 12 µg/mL of both solvent groups did not show the morphological alteration (data not shown). Meanwhile, cells in the DCM group (25 µg/mL) [Figure 2]B, but not in the HEX groups (25 µg/mL) [Figure 2]F, were shrunken. Compared with the untreated cells [Figure 2]A, cells treated with 50 µg/mL of DCM and HEX extracts were round and shrunken [Figure 2]C, G]. Noticeably, cells treated with propolis extracts (100 and 200 µg/mL) in both solvents showed the cell morphological changes [[Figure 2]D, E, H, I].
Figure 2: Morphological changes of human salivary gland were observed under inverted microscope. (A) Untreated group as a control, (B)–(E) dichloromethane (DCM) group, (F)–(I) hexane (HEX) group. Treated groups were cultured in different concentrations of propolis; scale bar = 50 µmThe HSG viability varies depending on the propolis concentrations and the types of solvent [Figure 3], which was shown by the MTT assay. Compared with the untreated cells, there is no statistical difference in the % cell viability of cells treated with 6.25 and 12.5 µg/mL of propolis in DCM and HEX. Interestingly, cells in the DCM group first showed a decrease in % cell viability at the 25 µg/mL with a significantly statistical difference. The reduction in % cell viability at the 50 µg/mL of DCM and HEX groups was observed. Apparently, the cell viability in the DCM and HEX groups gradually insignificantly declined with the dose-dependent manner. The IC50 of each extract is shown: DCM (42.93 ± 2.70) and HEX (61.30 ± 5.39) [Table 2].
Figure 3: MTT assay demonstrates the % cell viability of propolis extracts on human salivary gland. Values from three independent experiments (n = 3) are shown as the percentage of mean ± standard deviation. *P < 0.05 compared to the untreated group
Table 2: Values of half-maximum inhibiting concentration (IC50) of propolis extracted by different solvents on HSG cells evaluated by MTT and LDH assays were shownThe HSG toxicity varies depending on the propolis concentrations and the types of solvent [Figure 4]. As expected, the result of the LDH assay demonstrated the opposite trend compared with that of MTT assay. Compared with the untreated cells, there is no difference in % cytotoxicity among cells treated with low concentrations (6.25 and 12.5 µg/mL) of crude extracts. However, % cytotoxicity of cells in the DCM group exhibited a dose-dependent increase from 25 to 200 µg/mL. Cells in the HEX group at 50, 100, and 200 µg/mL presented approximately 60%–80% of cytotoxicity. The IC50 of each propolis extract is shown: DCM (34.94 ± 0.22) and HEX (42.32 ± 1.00) [Table 2].
Figure 4: Lactate dehydrogenase (LDH) assay demonstrates the % cytotoxicity of propolis extracts on human salivary gland. Values from three independent experiments (n = 3) are shown as the percentage of mean ± standard deviation. *P < 0.05 compared to the untreated group; PC = positive control (cells treated with the lysis buffer) Discussion This study is aimed to evaluate the cytotoxicity effect of propolis extracts by two different solvents, DCM and HEX, on HSG cell line using cell morphological changes, MTT and LDH assays. The HSG cell line, labeled as a submandibular ductal cell line, is commonly used as in vitro models to study for salivary gland research. However, this cell line has recently been considered as HeLa derived by short tandem repeat (STR)-DNA profiling[16] Nevertheless, the in vivo transplantation in mice showed these cells were able to generate a mass resembling salivary gland-like tumor.[17]
Compared with various concentrations of propolis extracts, 50, 100, and 200 µg/mL of DCM- and HEX-extracted propolis clearly demonstrated the cytotoxic effect on this cell line. However, the DCM group exhibited the superior outcome than the HEX group. The HSG demonstrated their morphological alteration, which was prominently observed in the high concentrations of propolis, 100 and 200 µg/mL. Nevertheless, HSG initially changed their morphology after treated with 25 µg/mL of propolis in the DCM group. Regarding IC50 of MTT and LDH assays, propolis extracts by both DCM and HEX were toxic to HSG cells, compared with the untreated control cells. Interestingly, we observed that the sensitivity of LDH assay was higher than that of MTT assay. Additionally, the DMSO toxicity to the cells might affect the interpretation of propolis’ anticancer property. Our experiment did not show any differences in % cell viability (data not shown) between untreated cells cultured in complete medium and complete medium plus 0.4% DMSO, indicating the nontoxicity of 0.4% DMSO. This suggests the anticancer effect due to the propolis composition.
The MTT assay demonstrated that each extract exhibited distinct viability of HSGs. The propolis extracts by DCM and HEX inhibited the HSG proliferation in different degrees. This may be due to different types and/or amounts of active compounds potentially extracted by each solvent. A previous study indicated that the anticancer chemical compositions of propolis were better extracted in the solvents with less polarity such as DCM, and with no polarity such as HEX, compared with water or ethanol, which are solvents with the high polarity.[18],[19] Our results are similar to the study by Teerasripreecha et al. that crude extracts obtained using DCM and HEX were able to inhibit the proliferation of several types of cancer cells.[20] However, our study first reported the effects of different solvent-extracted propolis on HSG cancer cell line.
To better understand the effect of various solvent-extracted propolis inducing the cell death, LDH assay was exploited on HSG cells. The amount of LDH in the culture is directly proportional to the number of damaged cells.[21] DCM- and HEX-extracted showed an increased LDH activity with dose-dependent manner. Noticeably, the DCM-extracted propolis at 25 µg/mL exhibited the superiority to enhance the HSG damage, compared with the HEX-extracted propolis at the same concentration. However, the cell death mechanism of propolis-treated salivary gland tumor cells would be further addressed.
Previous studies have reported variable chemical compositions of propolis depending on geographical sources of propolis. These may influence the difference in active components and biological activity of propolis.[22] Propolis from Europe, North America, and Asia had high flavonoid and phenolic contents.[23] In contrast, Brazilian propolis showed high terpenoid and coumaric acid.[24] Nevertheless, various sources of propolis have still mostly shown the anticancer effect. For example, Chinese propolis showed the significant anticancer effect on breast cancer cells.[25] Portuguese propolis had the cytotoxicity on lung cancer cells.[26] Unexpectedly, there is no research on the effect of propolis extracts on salivary gland tumor cells. Therefore, our study first demonstrated that Thai propolis extracts inhibited HSG proliferation and also induced cell death, suggesting its anticancer property on salivary gland tumor cells.
Propolis extracts by various solvents displayed a variety of active components.[27] The common solvents used for extraction are water, methanol, ethanol, chloroform, DCM, ether, and acetone.[23] A study by Teerasripreecha et al. has revealed that the crude DCM and HEX sequential extracts of propolis exhibited antiproliferative activities across several cancer cell lines.[20] Consequently, we chose DCM and HEX to directly dissolve the propolis and examine their anticancer effect on salivary gland cancer cell line. In our study, DCM-extracted propolis had the percentage of yield comparable to that of HEX-extracted propolis. However, propolis extracts by DCM showed higher flavonoid and total phenolic compounds than those by HEX. Our results are similar to Sambou et al. that the yield of propolis and total phenolic and flavonoid contents of DCM were higher than those of HEX.[28] This may explain why propolis in the DCM group exhibited lower IC50 than that in the HEX group.
Several pharmacological compounds in propolis and their anticancer mechanisms have been reported.[4],[7],[10],[29],[30] Flavonoid (e.g., quercetin, chrysin, naringenin) and total phenolic compounds (e.g. caffeic acid phenethyl ester [CAPE] and p-coumaric acid) from propolis have been shown to diminish the in vitro cytotoxicity in human tumor cells and tumors in animals.[31] There are accumulating reports that many flavonoids exert anticancer activity, and the molecular mechanisms responsible for this effect have been studying.[32] CAPE suppressed human oral cancer cell proliferation by modulating tumor suppressor genes and downregulating the oncogenes and survival via inhibiting Akt signaling.[33] CAPE also exhibited potent therapeutic activity by decreasing cyclooxygenase-2 (COX-2) expression and showed immunomodulation, resulting in a suppression of prostaglandin-2 synthesis in human oral epidermal carcinoma KB cells.[34] In addition, CAPE induced apoptosis by altering the pro- and antiapoptotic protein expression as well as kinase C modulation and inhibited tyrosine kinase signaling.[35] Cardanol and cardol, which are phenolic compounds, exhibited as cytotoxic constituents in Thai propolis.[20] Consequently, we conducted the phytochemical testing to demonstrate the presence of both flavonoid and total phenolic components. The result suggests that the ability of propolis to inhibit salivary gland cancer cell line may come from the differences between two extracts, giving us a hint why DCM extract seems better. Nevertheless, the active ingredient of propolis affecting the salivary gland tumor cells needs to be further explored.
Conclusion Based on the cell morphological observation, MTT and LDH assays, propolis extracts obtained using DCM and HEX exhibited the cytotoxic effect on HSG tumor cell line by affecting HSG viability and cell death in different manners depending on its concentrations.
Future scope/clinical significance
The solvent type and concentration may influence the type and different amounts of active components in propolis. The further study is essential to identify the biological effect of active components in extracts by different solvents in salivary gland tumor cells. This would be applied for a future research and therapeutic strategy in salivary gland cancer. For instance, animal models may be used to demonstrate the effect of propolis on the prevention and treatment of salivary gland lesions. As the prevention purpose, the animals should be medicated with propolis before creating the lesion in the salivary gland. As the therapeutic purpose, the animal would be medicated after the lesion generated. Our current research is the first preliminary study providing the knowledge to use propolis as a natural product of choice for salivary gland cancer prevention and therapy.
Acknowledgements
We would like to thank Prof. Emeritus Kenneth Izutsu (Oral Health Sciences, University of Washington, Seattle, WA, USA) for kindly providing us submandibular HSG cell line. We also thank Drs. Boolakorn Kanoksermsab, Panida Wiset, Paphawee Jetanon, and Puree Chaopanitchareon for certain data analysis. We deeply thank Mr. Prapan Supapakpattanna for research experiments. Lastly, we thank staffs in the Research Center, Faculty of Dentistry, Mahidol University, for providing us all research equipment.
Financial support and sponsorship
The research did not receive financial support from either governmental or non-governmental organization.
Conflicts of interest
There are no conflicts of interest to be declared.
Authors’ contributions
Jirattikarn Kaewmuangmoon: Concept, design, investigation, data analysis; Kanokwan Charoonpatrapong: Writing-review and editing, validation; Kajohnkiart Janebodin: Data analysis, writing original draft, review and editing, validation.
Ethical policy and institutional review board statement
Not applicable.
Patient declaration of consent
Not applicable.
Data availability statement
Not applicable.
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