Study on antioxidant activity and cytotoxicity of Aronia melanocarpa leaf tea extracts

Abstract

Aronia leaf tea, which is generally discarded after harvesting the fruit, was prepared using three different methods. Water extract from dried Aronia melanocarpa leaf tea (DALT), water extract from steamed Aronia melanocarpa leaf tea (SALT), water extract from roasted Aronia melanocarpa leaf tea (RALT) were manufactured and their functional ingredients were analyzed. The total polyphenol contents in the DALT, SALT, and RALT samples were 33.67 mg GAE/g, 57.79 mg GAE/g, and 53.16 mg GAE/g, respectively. The results from the ABTS radical scavenging activity and FRAP assays showed that there was significantly higher antioxidant activity in SALT and RALT samples than in DALT. The MTT assay revealed that the cytotoxicity of SALT and RALT samples against HeLa cells was higher than that of DALT. These results verified that the phytochemical components of aronia leaves changed based on its tea preparation methods and aronia leaf extracts contain bioactive compounds that have potential health benefits.

Introduction

Aronia (Aronia melanocarpa) is a genus of deciduous shrubs, the chokeberries in the family Rosaceae (Wu et al., 2004) native to North America and known as black chokeberry. Recently, studies have actively investigated the bioactivities of aronia fruit such as its antioxidant (Bermudez-Soto and Tomas-Barberan, 2004), anticancer (Lala et al., 2006), anti-inflammatory (Ohgami et al., 2005), antidiabetic (Badescu et al., 2014), and immunomodulatory (Gasiorowski et al., 2000) activities. Moreover, the aronia fruit contains anthocyanins, vitamin C, phenols, and flavonoid compounds (Bermudez-Soto and Tomas-Barberan, 2004).

Tea is a popular drink that is traditionally prepared using Camellia sinensis leaves. Tea helps relax the mind and body and exerts several pharmacological effects (Kim and Yook, 2018). It is often classified based on its manufacturing or components, such as nonfermented tea, semifermented tea, fermented tea, postfermented tea, or substitute tea. Tea made with leaves from plants other than C. sinensis is called substitute tea, and interest and consumption of substitute tea is increasing since there can be more variety in the flavor, and it can be produced to be more aromatic and palatable (Park and Jung, 2017). Substitute tea is reportedly prepared from the leaves of deodeok (Kim et al., 2015), Ojuk (Kim et al., 2012), guava (Lee et al., 2020), and ginger (Hwang et al., 2003). Moreover, this tea has been prepared from the leaves of A. melanocarpa; however, although aronia leaves are typically discarded during the tea production process, studies have suggested that they are rich in polyphenols, flavonoids, and chlorophylls that possess antioxidant effects (Kim et al., 2010). As a previous study on aronia leaves, antibacterial mechanisms of Aronia melanocarpa leaf extracts (Magdalena et al.,  2021), antioxidant and antimicrobial effects of aronia extract on beef patties (Babaoglu et al., 2022), and hypoglycemic, antioxidant and antineurodegenerative activities of chokeberry (Zdunić et al., 2020) have been reported. However, only a few studies have reported the physiological activity of aronia leaf constituents during tea production, drying (Kim and Yook, 2018), and roasting (Park and Jung, 2017); therefore, further studies are required to completely elucidate the functionality and bioactivity of aronia leaves. Meanwhile, studies about variety bioactivity of aronia leaf were not performed enough. During production, most of the aronia leaves used are typically discarded as byproducts, and they are known to contain several bioactive substances. Therefore, the functionality of aronia leaves, which possess value as a functional food and are economically feasible, requires further investigation. Nonfermented leaf teas are typically prepared by roasting, steaming, or drying. Studies regarding various leaf teas using different manufacturing methods have shown differences in components and physiological activities (Farooq and Sehgal, 2019; Hwang et al., 2003; Lim et al., 2007). Therefore, A. melanocarpa leaf tea prepared through drying, steaming, and roasting processes was extracted using water, and the composition of the functional ingredients as well as the antioxidant and anticancer activities of the extract were analyzed in the present study.

Materials and methods

Materials

Aronia melanocarpa plants were grown in a farm in Songgok-ri, Nam-myeon, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea, and leaves were collected immediately before the experimentation. 2,2-diphenyl-1picrylhydrazyl (DPPH), 2,2’-amino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), dimethyl sulfoxide (DMSO), l-ascorbic acid, Folin–Ciocalteu’s phenol reagent, gallic acid, and quercetin were purchased from Sigma Aldrich (Saint Louis, MO, USA), and fetal bovine serum (FBS), penicillin–streptomycin solution, and 0.25% trypsin EDTA were purchased from Gibco (Billings, MT, USA). Dulbecco’s Modified Eagle Medium with high glucose (DMEM/high glucose), Roswell Park Memorial Institute medium (RPMI) 1640, and L-glutamine were purchased from HyClone Co. (Logan, UT, USA).

Preparation of leaf tea

Aronia leaves were collected and tea was prepared under three conditions. Water extract from dried A. melanocarpa leaf tea (DALT) was prepared using aronia leaves dried at 80 °C for 12 h and extracted with water. That from steamed A. melanocarpa leaf tea (SALT) was prepared using leaves steamed at 100 °C for 3 min, manually rubbed for 5 min, and extracted with water. That from roasted A. melanocarpa leaf tea (RALT) was prepared using leaves roasted at 230 °C for 2 min and manually rubbed for 5 min, and extracted with water. Distilled water (800 mL) and aronia leaves (80 g) were used for each extract condition, and extraction was performed in a water bath at 60 °C along with shaking for 12 h. Following extraction, solutions were filtered through filter paper (Whatman No. 2, England) and then concentrated using a vacuum concentrator (Eyela, Tokyo Rikakikai Co., Japan). The concentrated extracts were lyophilized in a freeze dryer (VD-400 F, TAITEC Co., Japan), and resulting powders were stored at − 18 °C until experimentation.

Total polyphenol content

The total polyphenol content of the aronia leaf tea extracts was analyzed using the Folin–Ciocalteu’s method (Singleton and Rossi, 1965). Overall, 5 mL of distilled water and 0.5 mL of Folin–Ciocalteu’s phenol reagent were added to 1 mL of the extract and stirred. After 8 min, 10 mL of 7% Na₂CO₃ solution was added, distilled water was added to reach a total volume of 25 mL, and the mixture was placed in the dark for 2 h. The absorbance was measured at 750 nm using a UV/visible spectrophotometer (UV-1800, Shimadzu, Japan). Gallic acid was used as a standard to create a calibration curve, and the total polyphenol content was calculated. Three measurements were obtained, and their mean was calculated and expressed as gallic acid mg per g equivalent (mg GAE/g).

Total flavonoid content

The total flavonoid content of the aronia leaf tea extracts was measured following the method reported by Moreno et al. (2000). Overall, 1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate, and 4.3 mL of 80% ethanol were added to 0.5 mL of the extract, and samples were placed in the dark for 40 min. The absorbance was measured at 510 nm using a UV/visible spectrophotometer. Quercetin was used as a standard to create a calibration curve, and the total flavonoid content was calculated. Three measurements were obtained, and their mean was calculated and expressed as quercetin mg per g equivalent (mg QE/g).

Total vitamin C content

The total vitamin C content of the aronia leaf tea extracts was measured with a quantification method announced in the Korean Food Standards Codex 2020 that uses 2,4-dinitrophenyl hydrazine (DNPH) (Ministry of Food and Drug Safety, 2020). The tea extract sample and metaphosphoric acid-acetic acid were mixed together at a ratio of 1:1 and then dissolved in 100 mL of diluted metaphosphoric acid-acetic acid mixture solution. The mixture was centrifuged and the supernatant was collected. A total of 2–3 drops of indophenol solution were added to 2 mL of the supernatant, which turned the solution red, and 2 mL of metaphosphoric acid-thiourea was added, followed by 1 mL of DNPH. The reaction proceeded at 37 ℃ in a water bath for 3 h. After the reaction, the mixture was placed on ice and 5 mL of 85% H₂SO₄ solution was added slowly. After this addition, the mixture was left at 25 ℃ for 30 min, and then the absorbance was measured at 520 nm with a UV/visible spectrophotometer. The experiment was repeated three times, and a calibration curve was obtained using l-ascorbic acid as the standard material.

Determination of phenolic compounds

Phenolic compounds in the aronia leaf tea extracts including gallic acid, rutin, myricetin, quercetin, and caffeic acid were quantified using HPLC (Prominence, Shimadzu, Japan) with a Phenomenex C18 RP column (5 μm, 250 × 4.6 mm). Aronia leaf tea extract was dissolved in distilled water and filtered with a 0.45 μm syringe filter. The mobile phase contained 2% acetic acid in water (solution A) and 2% acetic acid in methanol (solution B) and was eluted in gradation. Gradient elution was performed from 0 to 20 min using 75% A and 25% B, 20 to 35 min using 50% A and 50% B, 35 to 50 min 30% A and 70%, 50 to 75 min 100% B. The flow rate was set to 1.0 mL/min, and the column temperature was set at 30 °C. Gallic acid was detected at 265 nm and caffeic acid was detected at 320 nm. Rutin, myricetin and quercetin were detected at 370 nm. Data were collected and analyzed with Shimadzu Class-VP version 6.14 SP1 software.

DPPH radical scavenging activity

The DPPH radical scavenging activity of the aronia leaf tea extract was measured following a method published by Blois (1958). Briefly, 2 mL of a 7.5 × 10^− 5 M DPPH solution was mixed with 1 mL of each concentration of sample and incubated at 37 ℃ for 30 min. The absorbance was measured at 517 nm using a UV/visible spectrophotometer. l-ascorbic acid was used as a control and the DPPH radical scavenging activity was calculated using equation and expressed as percentage. Measurements were taken three times and then expressed as a mean value.

$$\text{DPPH}\text{radical}\text{scavenging}\text{activity}(\%)=\frac{Ab{s}_{\text{control}}-Ab{s}_{\text{samples}}}{Ab{s}_{\text{control}}}\times 100$$

  

ABTS radical scavenging activity

The antioxidant activity of aronia tea leaf extracts was evaluated using the ABTS radical scavenging activity assay established by Re et al. (1999). Briefly, 7 mM ABTS and 2.45 mM potassium persulfate was mixed at a ratio of 1:1 and incubated at 30 °C for 12 h. This mixture was diluted to 0.7 mM at 413 nm using potassium phosphate buffer (5 mM, pH 7.4). Furthermore, 4 mL of the diluted ABTS mixture was added to 40 µL of each concentration of samples; the absorbance was measured at 413 nm immediately after a reaction time of 1 min. L-ascorbic acid was used as the control, and the ABTS radical scavenging activity was calculated using the following equation and expressed as a percentage. Measurements were obtained three times and then expressed as a mean value.

$$\mathrm{ABTS}\mathrm{radical}\mathrm{scavenging}\mathrm{activity}(\%)=\frac{Ab{s}_{\mathrm{control}}-Ab{s}_{\mathrm{samples}}}{Ab{s}_{\mathrm{control}}}\times 100$$

  

Ferric-reducing antioxidant power (FRAP) assay

FRAP assay was conducted as described by Benzie and Strain (1996) to evaluate antioxidant activity of aronia tea leaf extracts by measuring the reduction of ferric ions to ferrous ions. A total of 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl₃·6H₂O were added together at a ratio of 10:1:1 v/v/v to prepare the FRAP reagent. Then, 3.5 mL of this reagent was added to 0.5 mL of each concentration of the samples. The absorbance was measured at 593 nm using a UV/visible spectrophotometer immediately after adding the reagent, and absorbance was re-measured at the same wavelength after incubating the sample at 37 °C for 5 min. A calibration curve was prepared with FeSO₄·7H₂O as a standard and the FRAP value was expressed as FeSO₄·7H₂O eq µM. Measurements were obtained three times and then expressed as a mean value.

Cell culture

A549, HeLa, Hep3B, and MCF7 cell lines were obtained from the Korea Cell Line Bank (Seoul, Korea). RPMI 1640 was used for culturing A549, HeLa, and MCF7 cells, whereas DMEM was used for culturing Hep3B cells. RPMI 1640 and DMEM were prepared with 10% FBS and 1% penicillin–streptomycin and cell line was maintained in a CO₂ incubator (37 °C, 5% CO₂).

MTT assay

To confirm the growth inhibitory effect of aronia leaf tea extract on cancer cells, an MTT assay was performed as previously described (Carmichael et al., 1987). The cell lines were grown in the medium and were seeded in 96-well plates at a density of 5 × 10⁴ cells/mL. After incubation for 24 h at 37 °C in a 5% CO₂, 80 µL of medium was removed, and each sample was added to each well and incubated at 37 °C in a 5% CO₂ incubator for 48 h. Further, 20 µL of the 5 µg/mL MTT solution was added to each well and the plate was incubated for 4 h. The media containing the MTT reagent was removed and 150 µL of the DMSO: ethanol (1:1, v/v) solution was added to each well. The plate was placed on a shaking CO₂ incubator for 30 min and the absorbance was measured at 550 nm using an ELISA reader (Sunrise, Tecan, Switzerland). The growth inhibitory effect of aronia leaf tea extract on cancer cells was calculated using the following equation below and expressed as the cytotoxicity against cancer cells.

$$\text{Cytotoxicity}(\%)=\frac{Ab{s}_{\text{control}}-Ab{s}_{\text{samples}}}{Ab{s}_{\text{control}}}\times 100$$

  

Sulforhodamine B (SRB) assay

The SRB assay was employed to measure cytotoxicity and was based on a method by Doll and Peto (1981). Cancer cells were seeded in a 96-well plate at a concentration of 5 × 10⁴ cells/mL and incubated for 24 h. Following incubation, 100 µL of the sample was added to each well and incubated at 37 °C in 5% CO₂ for 48 h. Then, the medium was removed, and 100 µL of 10% trichloroacetic acid (TCA) was added to each well. Plates were incubated at 4 °C for 1 h, TCA was removed, and the plates were washed five times with sterile water and dried at room temperature. Further, 100 µL of 0.4% SRB dissolved in 1% acetic acid was added to each well. After 30 min, the plates were washed five times with 1% acetic acid and dried at room temperature. Later, 100 µL of 10 mM Tris buffer (pH 10.5) was added to each well, and the absorbance was measured at 540 nm using an ELISA reader. The SRB assay results were calculated and expressed using the same method as described for the MTT assay.

Statistical analysis

The experimental results were expressed as mean ± standard deviation values and were verified using analysis of variance and t-tests with the SPSS software, version 20.0 (IBM Corp., Armonk, NY, USA). A post hoc test was performed with Duncan’s multiple range test; p-value of < 0.05 indicated significance.

Results and discussion

Extraction yield, total polyphenol content, and total flavonoid content

Table 1 shows the extraction yields from DALT, SALT, and RALT samples, which were 8.80%, 6.90%, and 9.78%, respectively. The yield from SALT sample was lower than that from DALT and RALT samples because of leakage of some soluble substances during the steaming of aronia leaves. The total polyphenol contents of DALT, SALT, and RALT samples were 33.67, 57.79, and 53.16 mg GAE/g, respectively (Table 1), with the highest content being in SALT samples, followed by RALT and DALT samples. Previously, Park and Jung (2017) measured the polyphenol content of an ethanol extract of aronia leaves and reported 32.30 mg catechin equivalent (CE)/g in the raw aronia leaf extract and 37.96 mg CE/g after the samples were roasted 6 times. According to Yu et al. (2008a), the total polyphenol content of a fresh extract of mother chrysanthemum flower tea was 6.75 mg/g, which increased to 12.80 and 17.44 mg/g with increase in the number of roasting processes. Hong et al. (1998) measured the total polyphenol content of chicory roots and reported that the value increased with increase in the roasting temperature and time. Rahul et al. (2022) measured the flavonoid content of steamed mulberry bark and showed that compared with the untreated group, the polyphenol content of steamed mulberry bark increased. The findings of these previous studies were consistent with our results that compared with DALT sample, SALT and RALT samples exhibited higher total polyphenol contents when subjected to a relatively high heat treatment. According to Lim et al. (2005), the enzyme activity of polyphenol oxidase, a polyphenol-denaturing enzyme commonly found in plants, increases at temperatures between 10 °C and 40 ℃ but decreases with increasing temperatures. Lee et al. (1983) reported that the enzyme activity of polyphenol oxidase decreases more rapidly at a higher temperature. Therefore, we considered that the enzymatic activity of polyphenol oxidase in aronia leaf decreased because of high-temperature processing for tea preparation. In addition, the higher total polyphenol content in SALT and RALT samples compared with that in DALT sample might be attributed to the hand-rubbing process. Yu et al. (2008b) reported that hand-rubbing manipulation may have destroyed internal tissues, thereby rendering the elution of phenolic compounds easier during the tea preparation.

Table 1.

Extraction yields, total polyphenol, flavonoid, and vitamin C contents in the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf

Sample	Extraction yields (%, w/w)	Total polyphenols (mg GAE/g)a	Total flavonoids (mg QE/g)b	Total Vitamin C (mg/100 g)c
DALT	8.80 ± 0.32 A	33.67 ± 0.88 C	13.29 ± 0.66B	11.94 ± 0.53B
SALT	6.90 ± 0.83B	57.79 ± 1.20 A	19.30 ± 0.50 A	15.83 ± 0.32 A
RALT	9.78 ± 0.18 A	53.16 ± 1.03B	9.98 ± 0.29 C	6.72 ± 0.05 C

DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea

^amg of gallic acid equivalent/g of sample

^bmg of quercetin equivalent/g of sample

^cmg of l-ascorbic acid equivalent/100 g of sample

All values are presented as the average of three measurements with standard deviation

A–C denote values that are significant as determined by Duncan’s multiple range test (p < 0.05)

The total flavonoid contents of DALT, SALT, and RALT samples were 13.29, 19.30, and 9.98 mg QE/g, respectively (Table 1). Woo et al. (2010) reported that the total flavonoid contents of young Solidago virgaurea leaves decreased by roasting at 121 ℃. Lim et al. (2007) showed that the total flavonoid content of roasted mulberry leaf tea was reduced compared with that of dried mulberry leaf tea. These results indicate that high-temperature treatment can destroy the flavonoids. In the present study, the total flavonoid content of RALT sample treated at a relatively high temperature (230 ℃) was the lowest, which is consistent with results of a previous study. Therefore, further research is necessary to optimize leaf tea preparation to minimize flavonoid destruction. Compared with the results of Kim et al. (2012) that the total flavonoid contents of hot water extracts of Ojuk leaves, commercial green tea, and mate tea were 7.58, 2.96, and 8.14 mg QE/g, respectively, our results demonstrated that the total flavonoid contents of aronia leaf tea extracts (DALT, SALT, and RALT samples) were higher than those of other leaf teas.

Vitamin C content

Table 1 shows the total vitamin C (ascorbic acid) content of aronia leaf tea extracts that were quantified by the 2,4-DNPH assay. The total vitamin C content of DALT, SALT, and RALT samples was 11.94, 15.83, and 6.72 mg/100 g (Table 1), with the highest being in SALT samples, followed by DALT and RALT samples. High temperatures denature ascorbic acid oxidase, an enzyme that oxidizes vitamin C, and thus prevents its destruction. However, vitamin C is highly sensitive to temperature; therefore, it may be destroyed by high temperatures (Hwang et al., 2003). A study reported that vitamin C content in citron juice significantly decreased when the juice was heated to 90 °C, 95 °C, 97.5 °C, and 100 °C and with increase in the heating time (Kim, 1999). Similarly, in the present study, the total vitamin C content was lower in samples that were heated at a higher temperature. The heating temperature of RALT samples was 230 °C, which was higher than 80 and 100 °C for DALT and SALT samples. Although the total vitamin C content of SALT samples was higher than that of RALT samples, Hwang et al. (2003) reported that the total vitamin C content of roasted ginger leaf tea was higher than that of steamed ginger leaf tea. This can be attributed to different heating times and temperatures used in the leaf tea preparation. Further studies are required to elucidate the optimal temperature for tea production at which the ascorbic acid oxidase can be inactivated without destroying vitamin C.

Phenolic compounds

Table 2 shows the HPLC analysis of the phenolic compounds in DALT, SALT, and RALT samples. The amounts of phenolic compounds in DALT sample were 169.21, 15.97, 10.33, 3.33, and 2.53 mg/100 g of rutin, caffeic acid, myricetin, gallic acid, and quercetin, respectively. The amounts of phenolic compounds in SALT sample were 752.72, 85.10, 43.31, and 0.09 mg/100 g of rutin, caffeic acid, myricetin, and quercetin; gallic acid was not detected based on its standard curve. The amounts of phenolic compounds in RALT samples were 1064.90, 239.85, 104.51, and 1.37 mg/100 g of rutin, caffeic acid, myricetin, and quercetin; gallic acid was not detected based on its standard curve. Among the analyzed phenolic compounds, the rutin content was the highest in all aronia leaf tea extracts, implying that rutin is the major phenol component of aronia leaf tea. Moreover, RALT sample exhibited relatively high rutin, myricetin, and caffeic acid contents; these phenolic compounds could have increased the antioxidant activity of extracts. In previous studies, some tea extracts that are widely consumed, were found to be abundant in phenolic compounds and to exert strong antioxidant effects. Farooq and Sehgal (2019) reported the correlation between the antioxidant activity and phenolic contents in various infusions of green tea and Ocimum gratissimum. According to Jo et al. (2021), who studied the antioxidant activity of rose flowers, the increased antioxidant activities could be caused by the high amounts of phenolic compounds.

Table 2.

Phenolic compounds in the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf

Sample	Phenolic compound (mg/100 g)
	Gallic acid	Rutin	Myricetin	Quercetin	Caffeic acid
DALT	3.33	169.21	10.33	2.53	15.97
SALT	ND	752.72	43.31	0.09	85.10
RALT	ND	1064.90	104.51	1.37	239.85

DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea. ND None detected

DPPH radical scavenging activity

The results of the DPPH radical scavenging activity of DALT, SALT, and RALT samples are summarized in Fig. 1. The IC₅₀ values of the l-ascorbic acid control and DALT, RALT, and SALT samples were 27.18, 60.59, 31.12, and 29.03 µg/mL, respectively. The aronia leaf tea prepared in the present study was a nonfermented tea similar to green tea; a chemical transformation occurs during heat treatment inactivating the enzymes in the leaves (Kang, 2010). Additionally, the Maillard reaction, a nonenzymatic browning reaction, occurs upon heat treatment affecting the amino acids and reducing sugars in the leaves and producing an amino-carbonyl byproduct with antioxidant activity (Kang, 2010). A previous study demonstrated that the rate of the Maillard reaction indeed increased as the reaction temperature increased (Chun et al., 1986); this may have occurred in aronia leaves in the present study.

Fig. 1.

DPPH radical scavenging activity of the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf. DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea, A.A ascorbic acid. A–C denote values that are significantly different among different concentrations and a–d denote values that are significantly different among different samples at the same concentration as determined by Duncan’s multiple range test (p < 0.05)

In the present study, a strong negative correlation between total polyphenol content and IC₅₀ value of the extract was observed, consistent with the results of previous studies (Farooq & Sehgal, 2019; Jo et al., 2021). Therefore, total polyphenol content could have contributed to the high DPPH radical scavenging activity of the extracts. Because IC₅₀ values were lower in SALT and RALT samples than in DALT samples, a possible explanation is that the Maillard reaction proceeded faster at the temperatures used in the SALT and RALT sample preparation. The DPPH radical scavenging activity tended to increase as DALT, SALT, and RALT sample concentrations increased. At 200 µg/mL, DALT and SALT samples showed 86.81% and 81.76% DPPH radical scavenging activities, respectively, whereas RALT samples showed a higher DPPH radical scavenging activity of 90.41%. DPPH radical scavenging activity IC₅₀ values have been reported for the methanol extract of persimmon leaves (64.47%) (Hong et al., 2008), methanol extract of deodeok leaves at 500 µg/mL (81%) (Kim et al., 2015), ethanol extract of loquat leaves at 500 µg/mL (68.28%) (Hwang et al., 2010), and ethanol extract of blueberry roasted leaves at 500 µg/mL (91.87%) (Kim and Lee, 2014). Compared with the findings of previous studies of leaf extracts, our results provide evidence that aronia leaf tea extracts exhibit high DPPH radical scavenging activity even at low concentrations.

ABTS radical scavenging activity

The ABTS radical scavenging activity assay was employed to evaluate the antioxidant activity of DALT, SALT, and RALT samples (Fig. 2). The IC₅₀ values of the l-ascorbic acid control and DALT, SALT, and RALT samples were 6.7, 16.22, 9.86, and 11.08 µg/mL, respectively. As the DALT, SALT, and RALT sample concentrations increased, the ABTS radical scavenging activity tended to increase. All three samples showed excellent antioxidant activity; at 50 µg/mL, SALT, DALT, and RALT samples showed 97.10%, 96.33%, and 95.80% activity, respectively. These results indicated that the ABTS radical scavenging activity in all samples was relatively high compared with previously published results. The ABTS radical scavenging activities of an East Asian stripe maple leaf ethanol extract (Park et al., 2016) and a guava leaf ethanol extract (Lee et al., 2020) were reported to be 19.06% and 64.2%, respectively, at 100 µg/mL concentration, whereas those of a chloroform extract of apricot tree leaves and an ethanol extract of apricot tree leaves were 82.78% and 77.30%, respectively, at a 200 µg/mL concentration when apricot tree leaves were extracted by a solvent (Rho et al., 2015). Similar to the DPPH radical scavenging activity assay results, the antioxidant activities of SALT and RALT samples were higher than those of DALT samples; this can likely be because of the increased heating temperatures employed in SALT and RALT sample preparation compared with those used for DALT sample preparation, thereby causing an increase in the Maillard reaction rate. The ABTS radical scavenging activity assay results confirmed the high antioxidant activity of aronia leaf tea extracts even at lower concentrations than those previously reported.

Fig. 2.

ABTS radical scavenging activity of the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf. DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea, A.A ascorbic acid. A–D denote values that are significantly different among different concentrations and a–d denote values that are significantly different among different samples at the same concentration as determined by Duncan’s multiple range test (p < 0.05)

FRAP assay

Table 3 shows the FRAP values obtained from DALT, SALT, and RALT samples according to their concentrations. As the sample concentrations increased, the FRAP values also increased. At the highest concentration (200 µg/mL), the FeSO₄ eq µM of DALT, SALT, and RALT samples were 60.39, 109.53, and 142.10 µM, respectively. The FRAP values were the highest in RALT sample, followed by SALT and DALT samples. This finding is consistent with our previous suggestion that the Maillard reaction proceeds more rapidly at higher temperatures (i.e., at temperatures used during SALT and RALT sample preparation), resulting in higher amounts of amino-carbonyl products that possess radical scavenging activity. Polyphenol oxidase in pineapple inactivation reportedly occurs at 75 °C (Chutintrasri and Noomhorm, 2006). It was considered that polyphenol oxidase in aronia leaf was inactivated by roasting or steaming treatment and that the oxidation of phenolic compounds by the oxidative enzyme was inhibited, which could affect FRAP activity. Meanwhile, in the measurement of phenolic compound contents, the rutin content in RALT sample was considerably high, potentially increase FRAP activity. The result of Han et al. (2001), which studied the antioxidant activity of rutin isolated from the fruit of Prunus menu, showed that rutin exhibited high antioxidant activity.

Table 3.

Ferric reducing antioxidant power assay of the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf

Concentration (µg/mL)	FRAP values (FeSO4 eq µM)
	DALT	SALT	RALT
50	14.20 ± 0.25Dc	27.58 ± 0.10Db	37.94 ± 0.71Da
100	30.94 ± 0.71Cc	59.25 ± 0.14Cb	79.47 ± 2.41Ca
150	46.28 ± 1.66Bc	86.94 ± 3.01Bb	115.43 ± 3.45Ba
200	60.39 ± 3.94Ac	109.53 ± 3.95Ab	142.10 ± 1.13Aa

DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea

A–D denote values that are significantly different among different concentrations of the same sample as determined by Duncan’s multiple range test (p < 0.05)

a–d denote values that are significantly different among different samples at the same concentration as determined by Duncan’s multiple range test (p < 0.05)

Previously, Kim et al. (2011) reported that a FRAP assay was employed on the fruit and leaves of Ziziphus jujuba (jujube) that had been extracted with methanol and fractionated with chloroform, butanol, and distilled water. They found that the FRAP activity was higher in the solvent fraction of its leaves than in its fruit fraction. Another study reported that in aronia, the FRAP values were the highest in the leaves, followed by the stems and fruits. The results of the DPPH radical scavenging activity, ABTS radical scavenging activity, and FRAP assays indicate remarkably high antioxidant activity of aronia leaves and its potential value as a new functional food. Studies regarding the health benefits of other parts of the aronia plant may be warranted.

MTT assay

Figure 3 shows the results of the MTT cytotoxicity assay that tested aronia leaf tea extracts at 100, 200, 300, 400, and 500 µg/mL against cancer cell lines. In A549 cells, the cytotoxicity ranges were 12.88–42.95%, 25.56–88.46%, and 16.93–76.44% for DALT, SALT, and RALT samples, respectively. As the sample concentrations increased, their cytotoxicity increased. In HeLa cells, the cytotoxicity ranges were 26.10–64.47%, 35.93–89.14%, and 23.15–84.00% for DALT, SALT, and RALT samples, respectively. In Hep3B cells, the cytotoxicity ranges were 70.39–80.43%, 62.15–87.01%, and 38.01–65.45% for DALT, SALT, and RALT samples, respectively. In MCF7 cells, the cytotoxicity ranges were 38.20–70.23%, 47.61–84.48%, and 31.04–62.60% for DALT, SALT, and RALT samples, respectively. In a similar study, 500 µg/mL of a methanol extract of carrot leaf affected cell viability by 57% and 62.8% in HeLa and MCF7 cells, respectively (Shim et al., 2001). Our results show that the cytotoxicity of the water extract of aronia leaf in HeLa and MCF7 cells was higher than that of a methanol extract of carrot leaf. Notably, SALT and DALT samples were the most cytotoxic at 500 µg/mL in HeLa cells and affected their viability more strongly than they affected any other cell line. In addition, SALT samples exhibited high anticancer activity of more than 80% in all cancer cells evaluated at a concentration of 500 µg/mL. As previously mentioned, the high cytotoxicity of SALT is thought to be influenced by its high content of total polyphenol content.

Fig. 3.

Cytotoxicity of the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf on human cancer cell lines by MTT assay. DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea. A–E denote values that are significantly different among different concentrations in the same cells and a–d denote values that are significantly different among different cells at the same concentration as determined by Duncan’s multiple range test (p < 0.05)

SRB assay

An SRB assay was performed to investigate the cytotoxicity of aronia leaf tea extract samples against cancer cells at concentrations of 100, 200, 300, 400, and 500 µg/mL (Fig. 4). In A549 cells, the cytotoxicity ranges were 47.47–64.52%, 49.40–84.75%, and 44.47–58.07% for DALT, SALT, and RALT samples, respectively. As the sample concentrations increased, their cytotoxicity increased. The cytotoxicity ranges against HeLa cells were 28.62–49.95%, 13.71–48.29%, and 26.74–32.72% for DALT, SALT, and RALT samples, respectively. The cytotoxicity ranges against Hep3B cells were 57.09–66.53%, 51.60–81.43%, and 44.48–56.83% for DALT, SALT, and RALT samples, respectively. The cytotoxicity ranges against MCF7 cells were 55.28–68.77%, 57.81–76.97%, and 53.97–80.96% for DALT, SALT, and RALT samples, respectively. In the SRB assay, SALT showed typically high cytotoxicity against all examined cancer cell lines and exhibited relatively high cytotoxicity against A549 and Hep3B cells. Therefore, the high total polyphenol content was considered to cause high cytotoxicity against cancer cells. Polyphenol is reported to be effective in anticancer agents because phenolic rings inactivate carcinogenic substances by combining with free radicals that possess carcinogenic properties (Lewandowska et al., 2016).

Fig. 4.

Cytotoxicity of the water extracts of dried, steamed, and roasted Aronia melanocarpa leaf on human cancer cell lines by SRB assay. DALT water extract from dried Aronia melanocarpa leaf tea, SALT water extract from steamed Aronia melanocarpa leaf tea, RALT water extract from roasted Aronia melanocarpa leaf tea. A-D denote values that are significantly different among different concentrations in the same cells and a-d denote values that are significantly different among different cells at the same concentration as determined by Duncan's multiple range test (p < 0.05)

Meanwhile, a previous study that conducted a similar SRB assay with a 70% ethanol extraction of rhododendron leaves in Hep3B and MCF7 cells and reported the viability to be affected by 42.34% and 67.71% at 500 µg/mL, respectively (Byun et al., 2005). Our results showed that aronia leaf tea extracts exhibited higher cytotoxicity against the same cancer cell lines. Because aronia leaf tea extracts showed significant cytotoxicity against MCF7 cells, a more profound study should be conducted to determine the mechanism of cytotoxicity.

In the present study, the physiological activities of a domestic aronia leaf tea extract were evaluated. Water extracts of fresh aronia leaves were pretreated by drying, steaming, or roasting to prepare the study samples DALT, SALT, and RALT. The functional ingredients and antioxidant and anticancer activities of these samples were analyzed. The results showed that aronia leaves contain high contents of phytochemicals and functional components that exert antioxidant and anticancer activities, suggesting that these leaves should not be discarded during tea production. Further research is required to optimize aronia leaf tea production and to minimize the destruction of aronia leaf tea phytochemicals during pretreatment.

Declarations

Conflict of interest

The authors disclose the following: The authors declare no conflict of interest.