Antioxidant Properties of Aronia melanocarpa and Morinda citrifolia Juices and their Impact on Bladder Cancer Cell Lines

Abstract

Background

Aronia melanocarpa and Morinda citrifolia are valuable sources of bioactive compounds, mainly polyphenols, that provide health benefits. The antioxidants can scavenge free radicals that generate oxidative stress, resulting in various chronic diseases, including cancer. This research analyzed the in vitro effect of Aronia melanocarpa and Morinda citrifolia (Aronia and Noni) juices on urinary bladder cancer cells (T24) in comparison with normal uroepithelial cells (SV-HUC1).

Material/Methods

The study was preceded by analysis of the antioxidant properties of the tested juices – antioxidant capacity, total polyphenols, total flavonoids, and total anthocyanins. Moreover, the phenolic acids and selected flavonoids in the juices were determined through high-performance liquid chromatography.

The cytotoxicity of the tested juices was analyzed in cell lines via 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.

Results

Aronia and Noni juices affected the viability of both tested cell types in a time- and concentration-dependent manner. The results showed that Aronia and Noni samples were significantly more cytotoxic to urinary bladder cancer cells than to normal cells. Furthermore, it was found that Aronia juice was more cytotoxic against the tested cells than Noni juice. Aronia melanocarpa juice affected T24 cancer cells already at a concentration of 1.56% while Noni juice needed higher concentrations, starting at 12.5%. The greater efficacy of Aronia could be the result of its higher antioxidant capacity (629.4 vs 77.3 mg Tx/100 mL) or its higher content of total polyphenols (1036.6 mg vs 235.7 mg gallic acid equivalents/100 mL).

Conclusions

The results are promising and could indicate that Aronia and Noni juices exert beneficial effects against bladder cancer cells. Further studies should be performed, especially on other cancer cell lines.

Introduction

Chronic diseases and cancer are among the most common diseases in this day and age. Despite the development of new and improved treatment and diagnosis methods, cancer is still the second most common cause of deaths. Bladder cancer is among the most common malignant tumors in humans (more common in men). About 9 out of 10 people with this cancer are over the age of 55.

The treatment methods depend on the cancer stage in bladder cancer and include surgical procedures with either chemotherapy, radiotherapy, or newer types of medicines such as immunotherapy or targeted therapy [1,2]. Due to the insufficient effectiveness of currently used treatments, methods are sought that would complement the current therapy or would constitute an alternative approach in patients resistant to standard treatment [3,4]. Oxidative stress is also found in various cancer cells, and antioxidants have been regarded as having potential value in cancer chemotherapy [5]. Antioxidant plant extracts can be tested for their potential therapeutic effects on different cancer cells. Aronia berry extracts have been well documented for their potential therapeutic effects on different cancer cells, including human breast, cervical, colon, glioblastoma, liver, and lung cancer, as well as leukemia cells [5–8], but lack studies on bladder cancer cells.

The fruits of Aronia melanocarpa (common name: Aronia or chokeberry) are rich in numerous bioactive compounds, such as polyphenols (especially anthocyanins), as well as vitamins (vitamin C and vitamin E), minerals (potassium, calcium, and magnesium), organic acids, and pectins [9]. The presence of many phytochemicals and the high antioxidant capacity of chokeberry fruits predispose them to being used in treatment of chronic diseases related to oxidative stress, especially diabetes, cardiovascular diseases, and cancer [9,10]. Several mechanisms of action have been identified as being involved in the chemopreventive effect of polyphenolic extract of Aronia melanocarpa fruit, such as prevention of oxidation, reduction of oxidative stress, induction of detoxication enzymes, induction of cell cycle arrest apoptosis, regulation of the host immune system, anti-inflammatory activity, and changes in cellular signaling [9,11], thereby implicating a greater array of use for these fruits than for other berries. Taking into account its considerable content of bioactive compounds with high antioxidant properties and the related health potential, Aronia melanocarpa is considered a superfruit in Europe [12].

Another superfruit is the Noni fruit (Morinda citrifolia L.), originating from Southeast Asia and grown in South America and the Caribbean Islands. Noni juice is characterized by a considerable content of vitamin C (53.2 mg/100 mL) and potassium (150 mg/mL) and many other bioactive compounds with antioxidant properties [13,14]. Numerous phenolic compounds have been identified in Morinda citrifolia L., mainly iridoids and flavonoids, such as rutin and scopoletin [15]. Recently, Noni juice has been found to contain some quantities of phenolic acids (mainly ferulic and caffeic acids) and resveratrol [16]. The bioactive compounds contained in Morinda citrifolia L. juice endow it with health-promoting properties contributing towards the prevention of cardiovascular diseases and cancer.

A small number of in vitro studies and tests on cell lines performed so far have shown that Aronia and Noni juices may be effective in inhibiting the development of certain types of cancer. Recent studies have tested the impact of Aronia juice on the growth of human breast, leukemia, colon, and cervical tumor cell lines; and Noni juice on breast, kidney, and lung cancer cell lines. The obtained results showed that the mechanism of action of these juices in reducing cancer cell line viability relies on apoptosis induction, cell cycle arrest, oxidative stress reduction, and cell invasion and migration inhibition via regulating the AKT/NF-κB signaling pathway [9,17–22]. However, there is a lack of other studies concerning the effectiveness of Aronia and Noni juices on various other cancer cell lines, including bladder cancer cell lines.

Currently, it is known that the therapeutic effect of plant extracts is related to the presence of bioactive compounds, especially polyphenols. Numerous studies have demonstrated that extraction of a single compound is of limited clinical utility. Better effects have been demonstrated by extracts and juices than by singly isolated compounds because of the synergistic effect of various natural ingredients [21].

It seems that chokeberry and Noni juices, owing to their content of bioactive compounds, may support existing medical procedures in the treatment of cancer. Therefore, the aim of the present study was to conduct an analysis of the antioxidant properties of Aronia melanocarpa and Morinda citrifolia juices and conduct in vitro tests of their cytotoxic effect on urinary bladder cancer cells (T24) in comparison to normal uroepithelial cells (SV-HUC1). Given the lack of scientific reports concerning the use of chokeberry and Noni juices on bladder cancer cell lines, this research may be important. This study will answer the question of whether both tested juices are able to reduce bladder cancer cell viability, which will be the first step in designing further studies on this topic.

Material and Methods

Material

The study used organic Aronia melanocarpa juices (samples: Aronia 1 and Aronia 2), produced by traditional methods from Aronia fruits – both from Aronia melanocarpa (Michx.) Elliott cultivar (black chokeberry). These juices were selected on the basis of previous research concerning the antioxidant properties [23,24] and health aspects [25–27] of various fruit juices. Both juices were produced and supplied by a local manufacturer. Aronia melanocarpa fruits originated from an ecological plantation located in the south-western area of Poland, with an organic farming certificate according to article 29(1) of Regulation (EC) No 834/2007. The fruits were collected between September and October. The juices were produced by traditional methods, using wicker hydraulic presses and mild flow pasteurization (temperature no more than 80°C). The fruit and production process were carried out in accordance with the requirements of the eco certificate and without the use of any chemical substances. The second juice used for study was Noni juice (Morinda citrifolia L.). The organic Noni juice we used came from French Polynesia. This Noni juice was cold-pressed from whole fruits and underwent mild flow pasteurization (the temperature did not exceed 80°C) to preserve nutrients. All of the juice was naturally turbid and free from any additives. All of the juices were tested for phenolic compounds [total polyphenol content, Fast Blue BB assay (FBBB), total flavonoid content, and total anthocyanins] and antioxidant capacity [tested with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays]. High-performance liquid chromatography (HPLC) analysis was used to identify phenolic acids and flavonoids.

Methods

Total Polyphenol Content

The total polyphenol content of the samples was determined using the Folin–Ciocalteu assay (Sigma-Aldrich) [28]. First, 0.3 mL of a sample was placed in a 10-mL capacity tube. Next, 0.05 mL 2 mol/L Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 mL 20% sodium carbonate solution were added. The mixture was diluted by the addition of 4.15 mL distilled water and mixed. The absorbance was measured on a Rayleigh UV-1800 V/VIS spectrophotometer at 765 nm after 30 min incubation in the dark at room temperature. A calibration curve was performed with gallic acid. The results were expressed as milligrams of gallic acid equivalents per 100 mL of sample (mg GAE/100 mL) [29].

Fast Blue BB Assay

The Fast Blue BB (FBBB) assay is a novel method described by Medina [30] to quantify phenolic compounds through direct interaction of polyphenols with the Fast Blue BB reagent (4-benzoylamino-2,5-diethoxybenzenediazonium chloride hemi(zinc chloride) salt; Sigma-Aldrich) in an alkaline medium. A 0.2 mL aliquot of 0.1% Fast Blue BB reagent was added to 2 mL of samples and mixed for 1 min, and 0.2 mL 5% sodium hydroxide was added. The absorbance was measured on a Rayleigh UV-1800 V/VIS spectrophotometer at 420 nm after 90 min of incubation in the dark at room temperature. The results are expressed as gallic acid equivalents per 100 mL of sample (mg GAE/100 mL) [29]. The FBBB method demonstrates higher values of gallic acid equivalents than the Folin-Ciocalteu assay does [30,31]. Kang et al [32], who were the first to compare both methods, concluded that the results using the Folin-Ciocalteu assay may not be accurate because fruits, vegetables, and juices contain such ingredients as glucose, fructose, carotenoids, or ascorbic acid, which may distort the measurements of total polyphenol content [32].

Total Flavonoid Content

The total flavonoid content was measured using the colorimetric assay developed by Kapci et al [33]. Briefly, 0.3 mL of 5% sodium nitrite was added to 1 mL of sample at zero time. After 5 min, 0.3 mL of 10% aluminum chloride was added. At 6 min, 2 mL of 1M sodium hydroxide was added. The mixture was diluted by addition of 2.4 mL distilled water and mixed. The absorbance was measured on a Rayleigh UV-1800 V/VIS spectrophotometer at 510 nm. The total flavonoid content was determined by a (+)-catechin (Sigma-Aldrich) standard curve and was expressed as milligrams of catechin equivalents per 100 mL of sample (mg CAE/100 mL) [29].

Total Anthocyanins

Total anthocyanins were determined by the pH differential method of the Association of Official Analytical Chemists (AOAC; 2005.02) [34]. Juices were diluted according to appropriate dilution ratios (1 part sample and 4 parts buffer) by adding both 0.025 mol/L KCl (pH 1.0) or 0.4 mol/L CH₃COONa·3H₂O (pH 4.5) buffer solutions (Avantor Performance Materials). Samples were mixed and left in the dark for 30 min. Absorbance was measured on a Rayleigh UV-1800 V/VIS spectrophotometer at 520 nm and 700 nm, and the results were calculated using the following formula:

$$\text{A}=[{({\text{A}}_{520}-{\text{A}}_{700})}_{\text{pH}1.0}-{({\text{A}}_{520}-{\text{A}}_{700})}_{\text{pH}4.5}]$$

where A₅₂₀ is the absorbance measured at 520 nm and A₇₀₀ is the absorbance measured at 700 nm, at pH 1.0 and 4.5, respectively.

Total anthocyanins were expressed as milligrams of cyanidin-3-mono-glucoside equivalents (CGE) per 100 mL of sample (mg CGE/100 mL juice [34]; molar extinction coefficient=26.900 L/mol/mL and molecular weight=449.2 g/mol).

DPPH Assay

The antioxidant capacity of the fruit juices was determined by a modified Yen and Chen method, using 0.1 mmol/L methanol solution of DPPH (Sigma-Aldrich, St. Louis, MO, USA) [35]. The procedure was as follows: 0.1 mL of a sample was added to 2.9 mL of DPPH solution and mixed. The absorbance was measured on a Rayleigh UV-1800 V/VIS spectrophotometer at 517 nm after 30 min of incubation at room temperature in the dark. For each juice, samples were analyzed in 3 replicates and the results were used to calculate an average value. The percentage of DPPH scavenging was calculated using the following equation [29]:

$$\%\hspace{0.17em}\text{scavenging}=[({\text{A}}_{\text{DPPH}}-{\text{A}}_{\text{juice}})/{\text{A}}_{\text{DPPH}}]\times 100$$

where A_DPPH is the absorbance of the DPPH blank solution and A_juice is the absorbance of the sample solution.

The resultant value was then substituted into an equation of a previously prepared 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich) calibration curve. The antioxidant capacity of the samples was expressed as milligrams of Trolox equivalents (Sigma-Aldrich) per 100 mL of sample (mg Tx/100 mL).

ABTS Assay

The antioxidant capacity was determined by the Re et al [36] method with small modifications. In the ABTS method, ABTS (Sigma-Aldrich, St. Louis, MO, USA) and potassium persulfate solutions were mixed and stored overnight at room temperature in the dark for 12–16 h. The ABTS solution was diluted with methanol to an absorbance of 0.70±0.02 at 734 nm.

After the addition of 1.0 mL of diluted ABTS solution (A734 nm=0.700±0.020) to 0.01 mL of antioxidant compounds or Trolox standards in methanol, the absorbance was measured on a Rayleigh UV-1800 V/VIS spectrophotometer at 734 nm against methanol after 1 min. Quantification was performed using a Trolox standard curve. The antioxidant capacity of the samples was expressed as milligrams of Trolox equivalents (Sigma-Aldrich) per 100 mL of sample (mg Tx/100 mL) [29].

HPLC Analysis of Phenolic Acids and Flavonoids

The content of phenolic acids and selected flavonoids was determined by HPLC using the methods described by Krygier et al [37] and Hertog et al [38]. The analyses were performed using a Dionex LC system equipped with a photodiode array detector (Dionex) and the absorption spectra were recorded in the range of 200–600 nm. The flow rate was 1 mL/min, the column temperature was 30°C and the injection volume was 20 μl. Qualitative identification was done by comparing the retention times and spectra with the standards, which ensure better reliability of analysis than the percentage of probability of the compound identification. Simultaneous monitoring was performed at 280 nm for phenolic acids and 360 nm for flavonoids.

Phenolic acids were determined according the method described by Krygier et al [37]. The separation was performed on an Ascentis (Supelco) C18 column (250×4.6 mm; 5 μm). The binary mobile phase consisted of 0.1% (v/v) formic acid in methanol (eluent A) and methanol-acetonitrile (80: 20, v/v; eluent B).

Flavonoids were determined by a modified Hertog et al [38] method, after acidic hydrolysis. The separation was performed on an Ascentis (Supelco) C18 column (250×4.6 mm; 5 μm). The binary mobile phase consisted of 0.1% (v/v) formic acid in water-methanol (75: 25, v/v, pH 2.7; eluent A) and 0.1% (v/v) formic acid in methanol (eluent B).

Cell Lines

The human urinary bladder cancer cell line (T24) and human urothelium cell line (SV-HUC-1) were purchased from the American Tissue Culture Collection (ATCC; USA). The T24 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) Ham’s F-12 50/50 1X Mix (Corning, USA), and SV-HUC-1 cells in Kaighn’s Modification of Ham’s F-12 1X Medium (Corning, USA). Both culture media were supplemented with 10% fetal bovine serum (FBS) and 3 antibiotics (100 U/mL): penicillin/streptomycin and amphotericin B (Corning, USA). Cells were cultured in an incubator, at 37°C and 5% CO₂. The medium was changed every 2–3 days. After reaching 70% confluence, cells were passaged. For this purpose, the culture medium was removed, cells were washed 2 times with 1X phosphate-buffered saline (PBS); next, trypsin buffered with 0.2 mg/mL ethylenediaminetetraacetic acid (EDTA) was added; 0.05% for T24 and 0.25% for SV-HUC-1. Cells with enzyme were placed in an incubator (37°C and 5% CO₂) for 5 min; cell detachment was monitored under an inverted light microscope (DMi1; Leica, Germany). Next, the trypsin was inactivated by adding an equal volume of complete culture medium. The cells were centrifuged (Sorvall XTR, ThermoFisher Scientific, Germany) at 300× g for 5 min, and the cell pellet was suspended in fresh culture medium. The number of cells was calculated by mixing the cell suspension with an equal volume of 0.4% (w/v) trypan blue in PBS (Corning, USA) using a Neubauer chamber and the equation:

$$\frac{A}{4}·B·2·{10}^4$$

where:

• A = the number of calculated cells

• B = the volume of medium in which the cells were suspended

Cells from the third passage after cell line thawing were used in the experiment.

Cytotoxicity Assay

Cells were seeded on clear-bottom 96-well plates, 2.5×10³ T24 cells, and 12.5×10³ SV-HUC-1 cells were seeded per well. Next, cells were cultured in an incubator for 24 h; after that, different concentrations of the Noni (0.39%, 0.79%, 1.56%, 3.125%, 6.25%, 12.5%, 17%, and 25%) and Aronia (0.39%, 0.79%, 1.56%, 3.125%, and 6.25%) juice samples were added to the wells. The tested concentrations were established based on preliminary results performed on broader concentration ranges of 25% to 0.39% (data not shown). Based on the obtained results, the tested concentrations were established. Because, in the preliminary study, we observed a difference in efficacy between the 2 juices (Aronia juice was more effective than Noni juice), we used different concentrations for each juice. Both juices were filtered through a 0.22 μm filter before use. Working solutions were prepared by dilution of juices in the appropriate culture medium. The pH of the highest tested concentration of each juice was analyzed (Five Easy pHF20; Mettler-Toledo, Switzerland). Analysis of pH showed no influence of the Aronia and Noni juices on the pH value of the tested solutions. As a control, a fresh culture medium was used. Wells without cells were used as blanks. Cells were incubated with the tested compounds for 24 h; after that, the working solutions were removed, and each well was flushed 2 times with PBS. Then, 100 μl of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) solution at a concentration of 1 mg/mL was added. Cells were incubated at 37°C for 2 h. After that, the MTT solution was removed, and the formazan crystals that had formed were dissolved in dimethylsulfoxide (DMSO). The absorbance was measured at 570 nm and 655 nm using a Varioscan LUX plate reader (ThermoFisher Scientific, USA). Eight wells were used for each tested concentration. The experiments were repeated 3 times. The results obtained from the MTT assay was used for calculation of lethal concentrations (LC) values, defined as causing death of 10% (LC10), 50% (LC50), and 90% (LC90) of cells. Cell morphology was analyzed using a Lecia DMi1 inverted light microscope (Leica, Germany).

Statistical Analysis

The results of the analysis of antioxidant properties and polyphenol structures were statistically analyzed by calculating mean and standard deviation. The interpretation of the results was performed with MS Excel Analysis ToolPak software, one-way analysis of variance (ANOVA) using Tukey’s post-hoc test: different letters in the same row or column indicate statistical significance (defined as P≤0.05). In the section concerning in vitro studies, the average cell viability was expressed as a percentage relative to the control. All data were presented as mean±SD. The normal distribution of data was analyzed using the Shapiro-Wilk test. Parametric analysis was performed with one-way ANOVA with Tukey’s post-hoc test (GraphPad Prism 8, USA).

Results

Antioxidant Properties

Aronia 1 and Aronia 2 juices had significantly higher (P≤0.05) total polyphenol content than Noni juice (≡1000 mg GAE/100 mL vs ≡235 mg GAE/100 mL) (Table 1). These results were also supported by the other analytical method applied (FBBB assay). Aronia 1 juice had slightly more phenolic compounds than Aronia 2 juice, but the differences were not statistically significant (P≤0.05). The analysis showed that Noni juice had small amounts of flavonoids and virtually no anthocyanins. In contrast, both Aronia 1 and Aronia 2 juices had high contents of flavonoids (358.4–376.1 mg CAE/100 mL) and anthocyanins (298.4–328.4 mg CGE/100 mL) which determined their high antioxidant capacity (by both DPPH and ABTS assay).

Table 1.

Antioxidant properties of the analyzed fruit juices.

	Aronia 1	Aronia 2	Noni
Phenolic compounds
TP [mg GAE/100 ml]	1036.6±21.9a	984.4±30.6a	235.7±13.5b
FBBB [mg GAE/100 ml]	1751.1±7.2a	1674.0±35.6a	149.6±3.5b
TF [mg CAE/100 ml]	376.1±12.6a	358.4±14.5a	5.5±0.1b
TA [mg CGE/100 ml]	298.4±6.8a	328.4±8.9a	<0.1±0.0b
Antioxidant capacity
DPPH [mg Tx/100 ml]	629.4±33.7a	577.9±18.5a	77.3±0.6b
ABTS [mg Tx/100 ml]	891.2±12.0a	860.4±32.2a	139.0±2.7b

Data are mean±standard deviation (n=3). Statistical analysis was performed by one-way ANOVA using the Tukey’s post-hoc test: different superscript letters a, b in the same row indicate statistical significance (P≤0.05). TP – total polyphenol content; FBBB – 4-benzoylamino-2,5-diethoxybenzenediazo-nium chloride hemi(zinc chloride) salt; TF – total flavonoid content; TA – total anthocyanins; Tx – Trolox equivalents; GAE – gallic acid equivalents; CAE – catechin equivalents; CGE – cyanidin-3-mono-glucoside equivalents; DPPH – 1,1-diphenyl-2-picrylhydrazyl; ABTS – 2,2′-azino-di-[3ethylbenzthiazoline sulphonate].

The structures of the phenolic compounds in the samples were also examined (Table 2). In the group of free phenolic acids, chlorogenic acid was determined in the highest amounts, both in Aronia 1 and Aronia 2 juices, although it was not detected in Noni juice. Noni juice did not contain ferulic acid either, in contrast to the Aronia samples. Flavanols in Aronia 1 and Aronia 2 juices were represented mainly by epicatechins and smaller amounts of catechins. Noni juice was characterized by the presence of only catechins, but their amount was half that found in Aronia 1 juice. In the group of flavonols, quercetin was determined in Aronia 1 and 2, but undetected in the Noni sample. On the other hand, kaempferol was identified only in Noni juice, but not in the Aronia samples. Among the flavones, Aronia 1 and 2 had a higher content of apigenin than Noni juice, although the latter also contained luteolin. The results showed that the Aronia juices were characterized by a richer structure of phenolic compounds (slightly higher in the Aronia 1 sample). The Noni juice had a much lower amount of polyphenols, among which kaempferol and catechin were prevalent. Moreover, small amounts of resveratrol (stilbenes fraction) was identified in the Noni juice, but this compound was absent in Aronia 1 and Aronia 2.

Table 2.

HPLC analysis of selected phenolic compounds in juices (mg/L±SD).

Compounds	Aronia 1	Aronia 2	Noni
Free phenolic acids
– Gallic acid	tr.	0.35±0.03a	0.29±0.01a
– Chlorogenic acid	136.8±10.2 a	125.4±7.0 a	nd
– Caffeic acid	9.61±1.05b	14.3±1.0a	nd
– Coumaric acid	nd	nd	0.56±0.04
– Ferulic acid	0.49±0.05b	0.66±0.07a	nd
Bound phenolic acids *
– Gallic acid	tr.	tr.	nd
– Caffeic acid	440.0±15.0 a	385.4±3.5 a	0.85±0.15b
– Coumaric acid	19.0±0.8a	18.3±0.2a	tr.
– Ferulic acid	13.2±0.5a	10.1±0.1b	nd
Flavanols
– Catechin	4.2±0.2a	3.1±0.1b	1.9±0.2c
– Epicatechin	11.2±0.6 a	6.9±0.3 b	nd
Flavonols
– Myricetin	nd	nd	tr.
– Quercetin	13.8±0.3 a	12.5±0.3 a	tr.
– Kaempferol	nd	nd	1.9±0.1
Flavones
– Luteolin	nd	nd	0.4±0.1
– Apigenin	0.9±0.1 b	1.5±0.1 a	0.4±0.1c
Resveratrol	tr.	tr.	0.2±0.1

^*Bound phenolic acids=phenolic acids detected after alkaline hydrolysis - free phenolic acids.

LOD=0.1 mg/L; nd – not detected; tr. – trace. Data are mean±standard deviation (n=5). Statistical analysis was performed by one-way ANOVA using the Tukey’s post hoc test: different superscript letters a, b, c in the same row indicate statistical significance (p≤0.05).

The Aronia 1 and Aronia 2 juice samples, which were analyzed with beneficial results in the previous studies (details in Materials section), turned out to be similar to each other. Therefore, in the second part of the study on cell lines, we decided to use the Aronia 1 juice, because it was slightly better in terms of the content of bioactive compounds and antioxidant properties.

The high antioxidant capacity, high content of polyphenols, especially anthocyanins and chlorogenic and caffeic acid, in chokeberry juice may have a beneficial effect on cytotoxicity and reduced viability of bladder cancer cells.

The next stage of the study comprised tests to determine the effect of Aronia and Noni juices on bladder cancer cell lines (T24) in comparison with normal urothelial cells (SV-HUC-1).

In Vitro Studies

Aronia and Noni juices reduced the viability of both tested cell lines in a time-dependent manner (Figure 1). Aronia samples were more cytotoxic against tested cells than Noni samples, which was confirmed by the calculated LC values (Table 3). Significant changes in call viability were observed in 1.56% Aronia juice (63% viability) and in 12.5% Noni juice (45% viability). These values are consistent with the antioxidant results, indicating more potent antioxidant activity in the Aronia juice, with its higher phenolic and antioxidant compound content (Table 1). Comparing the effect against normal and cancer cell lines, statistical significance was observed only in the tests of Aronia melanocarpa juice in the higher tested concentrations of 1.56% and 3.125% (Figure 1). In this case, the lower LC concentrations were also calculated for cancer cells (0.40% vs 0.66% for LC10; 2.17% vs 3.04% for LC50; and 7.02% vs 9.56% for LC90), which indicated greater efficacy of this juice against cancer cells (Figure 1, Table 3). This effect was not seen in cells treated with Noni juice.

Figure 1.

Cytotoxic effect of Aronia and Noni juices on T24 and SV-HUC-1 cell lines. A decrease in viability with increasing juice concentration was observed. SV-HUC-1 – non-malignant human urothelium; T24 – human bladder cancer; **** P≤0.0001. Graphs were created using GraphPad Prism 8 v. 8.4.3 (GraphPad Software, LCC, USA), and figures were generated using Affinity Designer v. 1.10.6.1665 (Serif (Europe) Ltd., United Kingdom).

Table 3.

LC values calculated after treatment of T24 and SV-HUC-1 cell lines with Aronia and Noni juices. Values are presented as percentages.

Juice	Cell line	LC10	LC50	LC90
Aronia	T24	0.40	2.17	7.02
	SV-HUC-1	0.66	3.04	9.56
Noni	T24	1.4	11.9	22.5
	SV-HUC-1	1.7	12.3	23.0

Changes in the morphology of cells treated with Aronia juice (Figures 2A, 3A) were observed from a concentration of 1.56%, in which cells started to detach from the growth surface (more rounded cells visible) (Figures 2B, 3B). With the Aronia juice at a concentration of 3.125%, there was only a small number of attached cells remaining among the cancer cells, and no attached cells were visible at a concentration of 6.25% (Figure 2C, 2D). In the case of the normal cell line, even in the highest tested concentration, cells attached to the growth surface were visible (Figure 3C, 3D). In the case of Noni juice (Figures 4A, 5A), morphological changes were observed at a juice concentration of 12.5%; T24 cells became larger and more elongated (Figure 4B), and SV-HUC-1 cells started to lose their cell-to-cell contact (Figure 5B). At the 17% concentration, there was only a small number of attached cancer cells, and at a concentration of 25%, hardly any attached cells were visible (Figure 4C, 4D). In the same concentrations, normal uroepithelial cells started to detach from the growth surface in a sheet manner (Figure 5C); in the highest tested concentration, similar to the T24 cell line, only a small number of attached cells was visible (Figure 5D).

Figure 2.

Morphology of T24 cells after treatment with Aronia juice. Inverted light microscope, scale bar=200 μm. T24 – human bladder cancer cells. Pictures were created using LAS v.4.5.0 (Leica Microsystems, Switzerland), and figures were generated using Affinity Designer v. 1.10.6.1665 (Serif (Europe) Ltd., United Kingdom).

Figure 3.

Morphology of SV-HUC-1 cells after treatment with Aronia juice. Inverted light microscope, scale bar=200 μm. SV-HUC-1 – non-malignant human urothelium cells. Pictures were created using LAS v.4.5.0 (Leica Microsystems, Switzerland), and figures were generated using Affinity Designer v. 1.10.6.1665 (Serif (Europe) Ltd., United Kingdom).

Figure 4.

Morphology of T24 cells after treatment with Noni juice. Inverted light microscope, scale bar=200 μm. T24 – human bladder cancer cells. Pictures were created using LAS v.4.5.0 (Leica Microsystems, Switzerland), and figures were generated using Affinity Designer v. 1.10.6.1665 (Serif (Europe) Ltd., United Kingdom).

Figure 5.

Morphology of SV-HUC-1 cells after treatment with Noni juice. Inverted light microscope, scale bar=200 μm. SV-HUC-1 – non-malignant human urothelium cells. Pictures were created using LAS v.4.5.0 (Leica Microsystems, Switzerland), and figures were generated using Affinity Designer v. 1.10.6.1665 (Serif (Europe) Ltd., United Kingdom).

Discussion

During the past few years, Aronia melanocarpa (black chokeberry) has drawn attention owing to its particularly high amount of antioxidants. Aronia antioxidants are represented by polyphenols, such as phenolic acids, flavonoids (mainly flavanols, flavonols, and anthocyanins) and tannins [5,17,39–43]. Chokeberry fruit holds a remarkable position among other berries such as blueberries, red raspberries, red currant, strawberries, and blackberries [40–42]. The present study confirmed the high antioxidant properties of Aronia melanocarpa.

The total polyphenol content in chokeberry juices ranged from 984–1037 mg/100 mL, and this was similar to other studies (883–1109 mg/100 mL) [44], or slightly lower (1022–1795 mg/100 g) [39]. In these studies, chokeberry juices were a good source of anthocyanins (298–328 mg/100 mL) and the obtained results were similar to those from another study (284–686 mg/100 g [39]), or slightly higher (183–277 mg/100 g [44]). From earlier studies, it is known that the Aronia anthocyanin profile consists exclusively of cyanidin glycosides, and cyanidin-3-galactoside and cyanidin-3-arabinoside are the major representatives, accounting for more than 90% of the available anthocyanins in Aronia berries [17,23,39,43]. Aronia juice had a much higher content of polyphenols, including flavonoids and anthocyanins, than Noni juice, which resulted in higher antioxidant properties. The total polyphenol content in Noni juice was ca. 236 mg/100 mL and was much lower than the 748–770 mg/100g shown in other studies [45]. Also, the content of total flavonoids and anthocyanins in our research was lower than in the literature data [45]. The spectrum of phenolic compounds of Aronia melanocarpa juice was characterized by a high content of chlorogenic acid and caffeic acid. The high content of chlorogenic acid among the phenolic acids in our study is in agreement with other studies [39,43]. We also found flavanols (mainly epicatechin) and flavonols (mainly quercetin) in chokeberry juices, which were also seen in other studies [39,43]. Antioxidants in black chokeberry can scavenge free radicals, which are responsible for oxidative stress, which in turn leads to numerous chronic diseases, such as inflammation and atherosclerosis, as well as cancer and neurodegenerative diseases [8–10,26].

Some in vitro and in vivo studies have concluded that Noni fruits produce antioxidant, anti-inflammatory, anticancer, immunomodulatory, and anti-dementia effects [21]. Various bioactive compounds contained in Noni juice may be responsible for these benefits. In the present study, catechins were the dominant compounds in Noni juice, while in another study, in addition to catechins, rutin and quercetin were identified [46]. Application of phytochemical-rich plant extracts is being considered as a supportive treatment in cancer therapy, including therapy for bladder cancer.

Therefore, the present study investigated the effects of Aronia juice and Noni juice on T24 bladder cancer cells compared with normal SV-HUC-1 urothelial cells. The results showed that Aronia juice was more cytotoxic to T24 cancer cells than Noni juice. Aronia melanocarpa juice affected cancer cells starting at a concentration of 1.56%. However, Noni juice showed an effect on T24 cancer cells only at much higher concentrations, starting at 12.5% (Figure 1).

Until now, there were no data about the effectiveness of Aronia juice on bladder cancer cells, although the literature has reported the use of Aronia melanocarpa in the treatment of other types of cancer. In vitro experiments confirmed the impact of Aronia melanocarpa on the growth of human breast, leukemia, colon, and cervical tumor cell lines. Aronia berries show promising activity against human colon cancer cells [9]. Some commercial extracts of Aronia fruits, ie Aronia arbutifolia (red), Aronia prunifolia (purple), and Aronia melanocarpa (black), have been tested for their total phenolic content, antioxidant capacity, and growth inhibitory activity against HT-29 human colon cancer cells. The results revealed that only the extract of Aronia melanocarpa was active against HT-29 cells. This activity correlated with the antioxidant capacity, total phenolic content, and the levels of chlorogenic and caffeic acids [18]. Our studies confirmed the above observations. Aronia melanocarpa juice, rich in polyphenols, was effective against T24 bladder cancer cells. In the present study, chlorogenic and caffeic acid were also major bioactive compounds in chokeberry juice. The data presented by Stanisavljević et al [19] suggested that a large amount of chokeberry polyphenols undergo transformation during digestion; nevertheless, they are still potent as antioxidant and antiproliferative agents. The authors tested the effect of the digested juice on proliferation of Caco-2 cells and determined that the proliferative rate was reduced by about 25% [19].

Gąsiorowski et al [20] showed that anthocyanins from an extract of Aronia melanocarpa inhibited the mutagenic activity of benzo(a)pyrene and 2-amino fluorene in the Ames test. Moreover, they inhibited the generation and release of superoxide radicals by human granulocytes. These findings suggested that the antimutagenic effect of anthocyanins was exerted especially by their free radical scavenging activity, and by the inhibition of enzymes activating promutagens and converting mutagens to DNA-reacting derivatives. Other authors reported that extracts of Aronia melanocarpa may reduce oxidative stress in breast cancer patients before and after surgery and during various phases of oncology treatment [17]. Furthermore, Sharif et al [47] pointed to the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (7.15 g/L) in a test on an acute lymphoblastic leukemia Jurkat cell line. Their results showed that the juice inhibited cell proliferation, which was associated with cell cycle arrest in the G(2)/M phase, and caused the induction of apoptosis. Aronia juices used in the study were characterized by a slightly higher total polyphenol content (9.8–10.4 g/L). Rooprai et al [48] found greater effectiveness of chokeberry extract in the treatment of glioma compared with extracts of elderberry, blueberry, or citrus flavonoids.

It has been demonstrated that the major phenolic compounds of Aronia berries show potential anticancer activity. For example, anticancer potential of cyanidin-3-O-galactoside and chlorogenic acid and has been found. Cyanidin-3-O-galactoside was found to inhibit BGC-803 human gastric cancer cell growth through induction of cell apoptosis by various gene changes [5,49]. Chlorogenic acid acts on p53 and related proteins, as well as protein kinase and other targets to inhibit the proliferation, migration, and invasion of cancer cells [5,50].

Furthermore, Noni juice has also shown promise in in vitro studies, although it had lower antioxidant properties and a lower phenolic compound content than Aronia juice. Data from the literature has indicated that the health benefits of Noni juice were determined by other bioactive compounds than those in Aronia juice. Phytochemicals found in Noni juice include: iridoids and iridoid glycosides, anthraquinones and anthraquinone glycosides, lignans, neolignans, flavonol glycosides, damnacanthal, nordamnacanthal, scopoletin, morindone, alizarin, aucubin, and rubiadin [45,51,52]. Most of these compounds were not considered in the present study. Among the flavonoids, kaempferol and catechin were the main ones found in Noni juice. Moreover, 4 characteristic compounds, scopoletin, rutin, quercetin, and 5,15-dimethylmorindol (5,15-DMM), have been reported in all tested Noni fruits and commercial juices originating from different regions of the world [53].

Similar to Aronia juice, there is a lack of information in the literature about Noni’s effect on bladder cancer cells, and only a few studies have tested Noni on other cancer cell lines. Noni fruit fractions have been used on breast adenocarcinoma cell lines and a non-cancer cell line of human embryonic kidney [15]. The present study found that the effectiveness of Noni juice was poorer than that of Aronia juice. Moreover, there was no statistical difference between cancer cells (T24) and normal urothelial cells (SV-HUC1).

There is a lack of data from the literature concerning the use of Aronia melanocarpa and Morinda citrifolia juices on bladder cancer cells. It is reasonable to use plant extracts in studies on cell lines, rather than single bioactive compounds isolated from such extracts. For example, Potter [54] suggested that the metabolism of pure compounds not associated with the plant matrix is different from the metabolism of the same compounds found in fruits and vegetables. Nevertheless, the antiproliferative activity of isolated anthraquinones (1–8) against 5 human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7, and SW480, was evaluated by in vitro studies. Compounds 1–8 exhibited remarkable antiproliferative activities, which were comparable to those of doxorubicin [55]. Recently, a systematic review of 51 clinical and preclinical studies showed that Morinda citrifolia demonstrated various anticancer properties in different cancer models, via multiple mechanisms including antitumor, antiproliferative, pro-apoptotic, antiangiogenesis, antimigratory, anti-inflammatory, and immunomodulatory activities. The authors concluded that Noni is deemed to be a potentially valuable medicinal plant in the treatment of cancer through its many intrinsic pathways [56]. Various studies have revealed that individual compounds isolated from plant extracts often exhibit limited clinical usefulness because the synergistic effect of natural phytochemicals is lost in the process of isolation of such substances [21].

Based on our results and other authors’ data on other cancer cells, the mechanism of action of both juices should result directly from their composition. The presence of phenolic compounds in their composition can lead to apoptosis induction, cell cycle arrest, oxidative stress reduction, and inhibition of cell invasion and migration [9,17–22]. It should be noted that the effectiveness of juices can be different, depending on the origin of the fruits (latitude, time of year) [23]. Different cell lines can also influence cell viability; different bladder cancer cell lines (varying in grade) and clinical material isolated from patients may respond differently to tested juices. In order to better understand the effect of Aronia and Noni juices on bladder cancer cells, more experiments have to be performed, including in vitro 3D models, patient-derived organoids, and in vivo animal models.

Data in the literature have not ascertained the concentration of juices that can be achieved in serum or urine after oral administration. The main problem with such a calculation is that juices contain different bioactive compounds. The advantage of applying juices in bladder cancer treatment is that they can also be administered intravesically, directly after transurethral resection of bladder tumor. [2].

The present research has delivered promising results, indicating that both juices (especially Aronia melanocarpa juice) could be beneficial in the treatment of bladder cancer, although further studies should be performed.

Conclusions

The results indicate that Aronia melanocarpa juice is a rich source of bioactive compounds (mainly polyphenols) exhibiting high antioxidant properties. This was reflected in the significant efficacy (P≤0.0001 at a concentration of 1.56%) of the analyzed Aronia juice in an experiment on the T24 bladder cancer cell line, compared with normal SV-HUC1 urothelial cells. In turn, Noni juice had a lower total polyphenol content and lower antioxidant capacity than Aronia juice, and while it showed activity on the tested cancer cell line, it only occurred when the juice was given in higher concentrations compared with the Aronia melanocarpa juice. The studies showed that fruit juices are a complex matrix of bioactive compounds that may determine their influence on a bladder cancer cell line. Therefore, further research is needed on the effect of Aronia melanocarpa and Morinda citrifolia juices on various cancer cell lines to confirm their health-promoting effect. It may be useful to extend the research to include the use of juices on rat and human colon cancer cell lines.