Effects of Peel Extract from Citrus reticulata and Hesperidin, A Citrus Flavonoid, on Macrophage Cell Line

1. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia 2. Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia 3. Army Medical Center, Jl. Mayjen Soetoyo, Cililitan, East Jakarta, Jakarta 13640, Indonesia 4. Department of Histology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Jl. Farmako, Sekip Utara, Yogyakarta 55281, Indonesia 5. Faculty of Science and Technology, Universitas Aisyiah Yogyakarta, Jl. Ring Road Barat 63, Gamping, Sleman, Yogyakarta 55292, Indonesia


INTRODUCTION
Citrus plants (family: Rutaceae) have attracted researchers' attention due to their biological activities since the early 18 th century (Manthey et al., 2001). Their phytochemical healthpromoting properties are mainly based on the anti-oxidant activities of flavonoid compounds that contribute to the cardiovascular disease and cancer prevention, anti-inflammatory, antiviral, and antimicrobial properties of citrus (Barreca et al., 2017;Benavente-Garcia et al., 1997). Citrus species contain a variety of flavonoids, namely flavonones, 261 flavons, and flavonols (Benavente-García et al., 1997). Hesperidin, a flavonone glycoside, is a major flavonoid compound in Citrus reticulata (reviewed in Barreca et al., 2017). The first description of hesperidin by Lebreton in 1828 marked the beginning of researchers' vast interest in citrus flavonoids (reviewed in Manthey et al., 2001). As a fruit crop that is abundantly cultivated and consumed across the globe, citrus fruits generate a large amount of waste every year (Sharma et al., 2017). Citrus peels are one of the solid citrus waste products that need to be managed. Since 2007, the Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada (UGM), Indonesia has been exploring the extract of citrus peels in order to reveal its chemopreventive properties and, at the same time, to increase the utility of citrus waste (reviewed in Meiyanto et al., 2012). One of the studies was focused on the ethanolic extract of Citrus reticulata, commonly known as tangerine or mandarin, which has been shown to contain flavonoids (Armandari, 2010;Meiyanto et al., 2011) (Table I). In addition, hesperidin, as one of the main compounds, has been studied (Table I).
In vitro, it induces cell proliferation and angiogenesis in MCF-7 breast or WiDr colon cancer cells at concentrations of 10−1500μg/mL Puspita et al., 2008;Yunas et al., 2007) but can suppress MCF-7 cell
In vivo, 150-600μg Citrus extract shows antiangiogenic activities (Chrisnanto et al., 2008) and suppresses epithelial breast and hepatic cell proliferation in rats with chemically induced cancer at 750-1500mg/kg BW (Supriyati et al., 2008;Meiyanto et al., 2011). In the same manner as Citrus extract, hesperidin also displays differing properties depending on its concentration and the cell type. Hesperidin (5-200μM) increases cell viability in MCF-7 and WiDr cells but decreases cell viability more effectively in combination with an anticancer agent than the anticancer agent alone (Gilang et al., 2012;Hermawan et al., 2010). In the other cancer cell lines, T47D breast, HeLa cervix, and MCF-7 with HER2 over-expression (MCF-7 /HER2), hesperidin exhibits an IC50 value varying from 11 to 200μM (Febriansah et al., 2014;Kusharyanti et al., 2011;Setiawati et al., 2011). The effect of Citrus extract on MCF-7/HER2 is contrary to the estrogenic effect seen in ovariectomized mice (Adelina et al., 2015). In summary, Citrus extract and hesperidin may act differently depending on the concentration and the cell type. The dual or biphasic effect of these compounds on cell proliferation will influence whether the expected effect on cells is antiproliferative or proliferative. Therefore, the precise action of Citrus extract and citrus flavonoids need to be elucidated in various cell types. Besides the exploration of its anticancer potency, citrus and citrus flavonoids have also been studied for their anti-inflammatory activities (Manthey et al., 2001). Our group's work has been focused mainly on cancer cells, not on normal or inflammation-related cells (Table I). To our knowledge, no study has yet addressed the dual effect of citrus extract or citrus flavonoids on cell proliferation. Hence, an investigation of Citrus extract and citrus flavonoids on normal or inflammatory-related cells, i.e., macrophages, is important, especially considering the probability of their contradictory concentration-dependent effect.
In this study, we evaluate the effect of ethanolic extract from Citrus reticulata peel (Citrus extract) and hesperidin at various concentrations on the modulation of cell proliferation in the RAW 264.7 macrophage cell line. We reveal that both Citrus extract and hesperidin show a biphasic effect on RAW 264.7 macrophage cell viability. Additionally, Citrus extract at a moderate tested concentration can induce the expression of interleukin-10 (IL-10), an anti-inflammatory cytokine.

Preparation of Citrus extract
Citrus reticulata fruits were obtained from Kalisoro, Tawangmangu, Central Java, Indonesia in September and were identified in the Laboratory of Pharmacognosy, Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada (UGM). Healthy, green, mature but unripe fruits were washed, and the peels were collected and air dried without direct sunlight. Dried peels were powdered and macerated in 70% ethanol (Merck, Darmstadt, Germany) (10L for 1kg powder) for 5 days as previously described . The ethanolic fraction was separated and evaporated in a rotary vacuum evaporator until a brown viscous extract was obtained (yield was 3.21% (b/b)) (Armandari, 2010). The obtained extract was then identified by thin layer chromatography to detect flavonoid components as previously described . After citrobroric treatments, positive spots indicating flavonoids were developed under 245 and 366nm UV lights (Armandari, 2010). This extract is heretofore referred to as "Citrus extract."

Cell culture
The murine monocyte macrophage RAW 264.7 cell line was a gift from Prof. Tatsuo Takeya (Nara Institute of Science and Technology, Japan). Cells were cultured in Minimum Essential Media (Gibco, USA) supplemented with 10% (v/v) Newborn Calf Serum (NBCS) (Gibco, USA) and 2% (v/v) penicillin-streptomycin (Gibco, USA) in a 37ᵒC, 5% CO2 incubator and grown to confluence in 75cm 3 tissue culture flasks. After they reached 80% confluence, cells were scraped and used for experiments.

Cell viability assay
Cells (5 × 10 3 cells/well) were plated in 96well plates and cultured in a complete medium for 48h. The medium was then replaced with a medium containing various concentrations of Citrus extract or hesperidin and incubated for 24h. Cell viability was assessed by the MTT method as previously described (Ikawati et al., 2018) and carried out in at least triplicate for each experiment. Briefly, the absorbance at 595nm of diluted formazan after addition of 0.5mg/mL 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, Germany) in phosphate-buffered saline (PBS) followed by stopper solutions (10% SDS (Merck, Germany) in 0.1NHCl (Merck, Germany)) were measured in a microplate reader (Bio-Rad, Japan). The detailed step-by-step protocol is described in Armandari (2010). Untreated cells served as a control, while wells without cells served as a blank. The percent cell viability and the IC50 value were calculated as (absorbance of treated cells − absorbance of blank)/(absorbance of control − absorbance of blank) × 100% and by linear regression analysis as follows: cell viability (%, y axis) vs log concentration (μg/mL or μM, x axis) (Ikawati et al., 2018), including all tested dosage of the concentration series). Data are presented as the mean of two or three measurements per condition per experiment.

Immunostaining
Immunocytochemistry with an anti-IL-10 mouse monoclonal antibody (Dako) was carried out by the avidin-biotin complex method as previously described . Cells (1 x 10 5 cells/well) were plated on coverslips in a 24well plate until they reached 80% confluence. Cells were then incubated with Citrus extract at the designated concentrations for 15h. After removal of the medium, cells were washed with cold PBS, and then were fixed in cold methanol for 10min at -20ᵒC. The endogenous peroxidase activity was blocked with hydrogen peroxide, and nonspecific sites were blocked with normal goat serum (Novocastra), for 10min each at room temperature, followed by incubation in the primary antibody, anti-IL-10 (1:50), overnight at 4ᵒC. After washing with PBS, cells were incubated in IgG biotinylated universal secondary antibody (Novocastra) for 10min. Following washing, cells were incubated with streptavidin-peroxidase complex reagent (Novocastra) for 10min and then incubated with 3,3-diaminobenzinidine (DAB) substrate solution (Novocastra) for 2-10min to visualize the bound biotin. Cover slips were washed in distilled water and counterstained with Mayer's hematoxylin (Dako) for 1-3min. Cells were dehydrated in ethanol, cleared in xylene, and cover slips were mounted with a mounting medium. Protein expression was observed qualitatively with a light microscope (Olympus). A stained cover slip without primary antibodies served as a control.

Statistical analysis
Data are presented as mean ± standard deviation (SD) and were analyzed for significance using the Student's t-test. Values of p <0.05 were considered to indicate significance.

Higher, but not lower, concentrations of Citrus extract decrease RAW 264.7 cell viability
To examine the inhibition or induction of cell proliferation, RAW 264.7 cells were treated with a series of concentrations of Citrus extract, in the range from 1 to 1,000μg/mL, for 24h. As expected, lower concentrations of Citrus extract did not affect cell morphology. The cell morphology at lower concentrations (i.e., 10μg/mL and 50μg/mL) appeared the same as that of control cells ( Figure 1A). Moreover, the cell density increased after treatment with 50μg/mL extract. In contrast, at higher concentrations, starting at 500μg/mL, more cells appeared rounder and less flattened out in the well. These morphological observations imply a biphasic effect of Citrus extract.
After 24h of incubation with Citrus extract, cell viability was assayed by the MTT method. The higher concentrations of Citrus extract (500μg/mL, 750μg/mL, and 1000μg/mL) were able to decrease cell viability significantly (64%, 46%, and 36%, respectively, compared with 100% in the control without Citrus extract treatment) ( Figure 1B). The cell viability in the presence of 250μg/mL Citrus extract was 96%, while lower concentrations increased the cell viability, though not significantly, ranging from 108% to 138%. The highest cell viability was given by the lowest tested concentration, 1μg/mL. Thus, Citrus extract demonstrated a biphasic effect on RAW 264.6 macrophage cell proliferation.

Biphasic effects of hesperidin on RAW 264.7 cell viability
Hesperidin is one of the citrus flavonoids, a major compound in Citrus extract or citrus in general. Therefore, a similar cell viability assay was also carried out for hesperidin to determine 264 Volume 30 Issue 4 (2019) whether this compound would exert a similar biphasic effect to the extract. A series of concentrations varying from 1μM to 500μM (equal to 0.6μg/mL−305.3μg/mL) was used (Figure 2). The lowest tested concentration did not significantly affect cell viability. However, at 5−100μM, hesperidin increased cell viability significantly (116%−136% compared with 100% in the control without hesperidin treatment). At higher concentrations, hesperidin decreased cell  Effects of hesperidin on cell viability in RAW 264.7 macrophage cells. Cells were treated with a concentration series of hesperidin, incubated for 24h, and then assayed by MTT assay in triplicate. The graph of hesperidin treatment demonstrated a biphasic effect on cell viability. The experiments were carried out three times, and the graph represents means ± SD (n=3). Statistical significance was determined by means of the Student's t-test. Hash tags indicate significant increase ( # ; p<0.05) on cell viability compared with the control. Asterisks indicate a significant decrease (***; p<0.001 ****; p<0.0001) in cell viability compared with the control. viability by as much as 61% and 10% at 250μM and 500μM, respectively. These data confirmed the biphasic effect of Citrus extract on cell proliferation, and that effect was likely caused by its citrus flavonoid content.
Citrus extract has a lower IC50 value compared with hesperidin in RAW 264.7 cells IC50 values were calculated based on linear regression equations derived from the graph of concentration versus cell viability (Figure 3). The IC50 of Citrus extract and hesperidin was 756 g/mL and 203μg/mL (332μM), respectively, as expected ranging at the high tested concentration. The IC50 of Citrus extract was 3.7 times higher than that of hesperidin. This is plausible because Citrus extract may contain other compounds. Nevertheless, at the given IC50, studies on the utilization of Citrus extract rather than hesperidin may yield additional advantages, especially if the accessibility of a pure compound is limited. Figure 3. The IC50 value of Citrus extract or hesperidin in RAW264.7 macrophage cells. Cells were treated with a concentration series of Citrus extract (A) or hesperidin (B) for 24h and then assayed by the MTT method. Graphs of concentration versus percentage cell viability are presented as indicated. Points in A and B are presented as the mean of two or three experiments, respectively. The IC50 value was calculated by linear regression analysis as stated on each graph.

Citrus extract can induce IL-10 expression
Treatment with 250μg/mL Citrus extract induced expression of IL-10, an anti-inflammatory cytokine, indicated by a brownish color in the cytoplasmic area compared with control IgG (Figure 4, rightmost panel versus leftmost panel). However, at a concentration of 25μg/mL, Citrus extract did not affect IL-10 expression and appeared similar to cells without Citrus extract treatment (0μg/mL). Higher concentrations should be tested further to confirm whether the modulation of IL-10 expression caused by Citrus extract is also ensuing the biphasic trend. Figure 4. Effects of Citrus extract treatment on the expression of interleukin-10 (IL-10). Cells were incubated with Citrus extract at the indicated concentrations for 15h. Cells were immunostained for IL-10 (brown) and counterstained with Mayer's hematoxylin to visualize nuclei (blue). Staining without primary antibody served as a control (leftmost panel). Scale bar = 50μm.
Our findings exhibit that the ethanolic extract of Citrus reticulata (Citrus extract) and hesperidin, a citrus flavonoid, display a biphasic effect on the proliferation of a monocyte macrophage cell line, indicated by the cell viability parameter ( Figure 1B and 2). More importantly, the concentration of Citrus extract required to increase RAW 26.7 cell viability varied from 1μg/mL to 250μg/mL ( Figure 1B) compared with 10-1,500μg/mL in cancer cell lines ( Yunas et al., 2007;Ardiani et al., 2008;Puspita et al., 2008). Meanwhile, hesperidin at low concentrations (5−100μM) can increase cell viability (Figure 2), similarly in cancer cell lines (5-100 or 200μM) (Hermawan et al., 2010;Gilang et al., 2012). The IC50 value of hesperidin in the monocyte 266 Volume 30 Issue 4 (2019) macrophage cell line is 1.7-30 times higher than in cancer cell lines ( Figure 3B) (Kusharyanti et al., 2011;Setiawati et al., 2011;Febriansah et al., 2014). Taken together, the use of Citrus extract or hesperidin should carefully deliberate the concentration or dosage and the cell type.
Macrophages express interleukin-10 (IL-10) and usually triggered by inflammatory stimuli (Chung et al., 2007). Since citrus flavonoids has been studied extensively for their antiinflammatory activity (Manthey et al., 2001), it is plausible to observe IL-10 expression under Citrus extract or hesperidin treatments. Due to our limitations (Figure 4). At 250μg/mL, Citrus extract can induce IL-10 expression, but not at lower concentrations. In the future study, higher concentrations of Citrus extract should be tested to confirm whether the modulation of IL-10 expression caused by Citrus extract also follows the pattern of a dual effect. Furthermore, the expression of cyclooxygenase-2 (COX-2), one of the key enzymes in inflammation, was also tested. However, there was no COX-2 detected (data not shown). An inflammatory stimulus such as lipopolysaccharide (LPS) should be administered to the cells, similarly to the ones that have been reported previously (Kang et al., 2011;Sakata et al., 2003).
At lower concentrations, Citrus extract was able to maintain cell viability independently of the expression of the anti-inflammatory cytokine IL-10. Meanwhile, hesperidin at a lower concentration was able to promote cell proliferation. Therefore, a low concentration of Citrus extract and hesperidin is possibly useful for treating inflammatory diseases. On the other hand, application of a low concentration of Citrus extract or hesperidin should be avoided for cancer cells because it induces cell proliferation as previously reported in a colon cancer cell line by Ardiani et al. (2008). Alternatively, at higher concentrations, Citrus extract or hesperidin is beneficial to cancer cells due to their cytotoxic effects. Recently, genistein, another flavonoid found in soybean, a widely known potential chemopreventive agent, has been reported to demonstrate a biphasic mechanism on CHO-K1 cells: at low concentrations, it induces senescence and apoptosis in combination with estrogen, and at high concentrations, it modulates the cell cycle (Jenie et al., 2019). Hence, an investigation of the dose-dependent mechanism of Citrus extract and hesperidin should be carried out further.

CONCLUSION
Citrus extract and hesperidin exerted a biphasic effect on a macrophage cell line. The future development of Citrus extract as a co-chemotherapeutic, anticancer, or immunemodulatory agent should include careful consideration of its biphasic effect on each cell type.

ACKNOWLEDGMENT
This study was partly funded by "Hibah Penelitian Berkualitas Prima" (Prime Quality Research Grant) Faculty of Pharmacy UGM 2009 (granted to MI). The authors wish to state their contributions as follow: IA performed experiments for the Citrus extract; AK and YE performed cell viability assays for hesperidin; MI and IA wrote the manuscript and prepared the figures; MI oversaw all aspects of the study. All authors have read and approved the manuscript.