Physiochemical properties and neuroprotective function of Korean major yuzu varieties

Abstract

Three major yuzu varieties, Native, Namhae, and Tadanishiki in Korea were investigated on physiochemical properties and functional characterizations with respect to neuroprotective function (SH-SY5Y cells) or anti-inflammatory (Raw 264.7 cells). Among the three cultivars tested, Namhae had significantly higher fruit weight and total dietary fibers, while Tadanishiki had higher values of both total flavonoid and antioxidant activity. As functional compounds, Tadanishiki contained a 133% of hesperidin and 164% of naringin, compared to those of Native or Namhae. In comparison to other yuzu, Tadanishiki showed the higher cell viability, lower AChE content, lower protein levels of Bcl-2 /Bax, Caspase 3, and PARP at scopolamine-treated SH-SY5Y nerve cell. Tadanishiki and Namhae were effective for anti-inflammation by decreasing of NO production in LPS-induced Raw 264.7 cells. These results indicate that Tadanishiki yuzu, could be useful for application as a possible natural functional additive with high hesperidin content and excellent neuroprotective and anti-inflammation effects.

Keywords:
yuzu; variety; physicochemical property; neuroprotection; anti-inflammation

1 Introduction

Northeast Asian citrus plant known as yuzu (Citrus junos Sieb) is frequently used in traditional medicine (Kim et al., 2015). Higher amounts of phenolics and vitamin C are present in yuzu than in other citrus fruits. Citrus flavonoids, which mostly consist of flavanones, flavones, and polymethoxyflavones, exhibit antioxidant, anticarcinogenic, and anti-inflammatory bioactivities (Manthey et al., 2001; Yu et al., 2005). Yuzu is widely utilized in the food industry to make wines, jams, teas, juices, and a variety of other foodstuffs. Despite having richer levels of pectin and cellulose than other byproducts, such as yuzu peel and pomace, they are nevertheless discarded as waste after yuzu processing (Lee & Chang, 2020). Yuzu is distinguished from other citrus fruits by having a potent distinctive aroma and is well recognized for the delightful fragrance of its outer peel. Therefore, yuzu is used industrially in the production of sweets, beverages, cosmetics, fragrances, and aromatherapy products (Sawamura, 2010; Lan-Phi et al., 2009).

It is well known that the polyphenols and flavonoids found in natural antioxidants have potent antioxidant properties and the capacity to scavenge ROS, RNS, and free radicals (Chavez-Santiago et al., 2022). Hesperidin, naringin, and neohesperidine, the primary flavonoids of yuzu, have antioxidant action and can cross the blood-brain barrier. Thus, these citrus flavonoids may act to prevent neurodegeneration and enhance mental abilities (Youdim et al., 2003; Hirata et al., 2005; Marder et al., 2003). Especially, hesperidin and diosmin are known to have anti-inflammatory effects by preventing pro-inflammatory arachidonic acid derivates production (Benavente-García & Castillo, 2008; Manthey et al., 2001). In mice with scopolamine-induced amnesia, naringenin, has been shown to improve memory function and protect PC12 cells (Heo et al., 2004). Oral hesperidin administration decreases the severity of rat brain damage after stroke by lowering free radicals and inflammation (Raza et al., 2011).

According to several studies, relatively high amounts of antioxidant components like hesperidin or neohesperidine are mostly responsible for the yuzu's health advantages (Assefa et al., 2016; Donato et al., 2014). However, little information is available about neuroprotective or anti-inflammatory effect of yuzu. In the present study, we analyzed physicochemical characteristics, dietary fiber composition, and functional flavonoids by HPLC of three major yuzu varieties. Moreover, three yuzu varieties were compared to determine brain cell-protection, acetylcholinesterase (AChE) inhibition and Western blot on SH-SY5Y cells, and anti-inflammatory activity on Raw 264.7 cells.

2 Materials and methods

2.1 Reagents and materials

The yuzu fruits used in this study were grown at Jeonnam Agricultural Research & Extension Services’ fruit research facility (Wando, Jeonnam Province, Korea). Three varieties of yuzu such as Native, Tadanishiki and Namhae were harvested in November 2020. Three yuzu varieties were then sliced, freeze-dried (FD8512, Ilshin, Korea), and grinded by pulverizer (FM-681C, Hanil Electric., Korea). 50 mL of 80% methanol was used to extract the 1 g of freeze-dried material over a 3-hour period at 50 °C. DPPH, Folin-Denis reagent, gallic acid, quercetin, ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Naringin, narirutin, hesperidin, neohesperidin were purchased from ChromaDex (Irvine, CA, USA). The purity of all analytical reagents used for tests was at least 90%.

2.2 Physicochemical properties

Fresh yuzu from three different types were used to test weight, color, acidity, and soluble solids (Brix). A sample of 20 randomly selected yuzu fruits was taken and their weight was measured. The chromometer (CR-400 m KONICA. MINOLTA, Japan) was used to evaluate the chromaticity of the yuzu surface. 20 mL of distilled water (DW) was added to 2 g of sample in order to assess the soluble solid content (°Brix) and total acidity. The supernatant was then collected by centrifuging at 5000 g for 10 min (Combi 514R; Hanil Scientific Co. Ltd., Incheon, Korea). An electronic refractometer (ATAGO Co. Ltd., Tokyo, Japan) was used to measure the amount of soluble solids. By adding 2 to 3 drops of 0.1 percent phenolphthalein to 4 mL of supernatant and titrating the mixture to red with a 0.1 N NaOH solution, the acidity of the mixture was determined. The consumption capacity (mL) of the NaOH solution was determined and converted into citric acid.

2.3 Dietary fiber composition

Insoluble dietary fiber (IDF) and soluble dietary fiber (SDF): Dietary fiber were analyzed according to the method of Prosky et al. (1988). Samples were suspended in MES-TRIS buffer and sequentially digested with heat-stable α-amylase at 95-100 °C, protease at 60 °C, and amyloglucosidase at 60 °C. In tared fritted glass crucibles, digested enzymes were treated. Crucibles containing IDF were splashed with dilute alcohol and acetone and dried overnight in a 105 °C oven. To precipitate fibers soluble in the digests, filters and washing solution were mixed with four volumes of 95% ethanol. After 1 h, the precipitates were filtered through fritted glass crucibles. The residue's insoluble and water-soluble dietary fiber contents were added up to create the Total Dietary Fiber (TDF) value.

Acid detergent fiber (ADF) and neutral detergent fiber (NDF): Samples (0.5 g) were suspended in 200 mL of acid detergent solution (20 g of acetyl trimethyl ammonium bromide in 1 N H₂SO₄), 200 mL of neutral detergent solution (30 g of SDS, 18 .61 g of EDTA, 6.81 g of sodium borate, 4.56 g of disodium hydrogen phosphate) and 10 mL of 2-ethoxy ethanol and sequentially digested at 100 °C of 1 h. Crucibles containing dietary fiber were rinsed with dilute alcohol followed by acetone and dried overnight in a 105 °C oven (Equations 1-2).

$$Cellulose\left(mg/g\right)=ADF–Lignin/sampleweight$$ (1)

$$Hemicellulose\left(mg/g\right)=ADF–NDF/sampleweight$$ (2)

Lignin and pectin: To measure lignin, 0.5 g sample were suspended in 15 mL of 72% H₂SO₄ and stirred for 2 h. then diluted to the concentration of 3% H₂SO₄ and digested sequentially at 100 °C of 3 h. Dietary fiber-containing crucibles were rinsed with 150 mL of DW and dried for a whole night in an oven set to 105 °C. Using a pectin identification kit (Megazyme), the yuzu samples' pectin content was determined. Low ester pectin extracted from citrus peel was utilized as a standard for the calibration curve, which was used to determine the pectin content.

2.4 Total phenolic and flavonoid contents

The total phenolic content was calculated according to the Folin & Denis (1912) method. The 30 mL samples were diluted with 32.5 mL DW, 12.5 mL of Folin-Denis reagent was added, and the reaction was conducted for 6 min in complete darkness. Then, 12.5 μL of 7% sodium carbonate and 250 μL of DW were added to the mixed solution. A microplate spectrophotometer (Synergy HTX, Biotek Epoch, Agilent, Santa Clara, CA, USA) was used to detect the absorbance at 760 nm following 1 h incubation period. Flavonoid content was calculated from a calibration curve where quercetin was used as standard. 20 μL of sample were combined with 200 μL of diethylene glycol and 20 μL of 2 N sodium hydroxide. After reaction was conducted at 37 °C for 30 min, absorbance was read at 420 nm using microplate reader.

2.5 Functional phenolics by HPLC-DAD

The yuzu phenolic content was measured by our previously used method using a Agilent 1216 Infinity LC series system (Agilent Technologies, Palo Alto, CA, USA) (Jeon et al., 2022). The analysis was carried out by using the ZORBAX Eclipse Plus C18 column (4.6 x 250 mm, 5-Micron; Agilent Technologies), with mobile phases of 0.1% formic acid (solvent A) in DW and methanol-acetonitrile (solvent B). A two solvent gradient system was applied : starting with A:80, B:20 at 5-10 min; A:60, B:40 at 10.1-15 min; A:50, B:50 at 15.1-20 min; A:30, B:70 at 20.1-25 min; and A:0, B:100 at 25.1-30 min at flow rate of 0.5 mL/min, and the column temperature equal to 35 °C, with a 10 μL injection, the absorbance was detected at 280 nm.

2.6 DPPH or ABTS radical scavenging activity

Determination of the DPPH radical scavenging ability was carried out by the modified Blois (1958) method. The 50 μL samples were diluted constantly, 250 μL of 1 mM DPPH was added and followed by incubation at room temperature for 10 min. Absorbance was measured at 517 nm by microplate spectrophotometer with ascorbic acid used as a reference compound. DPPH radical scavenging activity was calculated as the following Equation 3:

$$\begin{array}{l}DPPHradicalscavengingactivity\left(\%\right)=\\ \left(1-sampleabsorbance/controlabsorbance\right)\times 100\end{array}$$ (3)

The ABTS radical cation (ABTS+·) was prepared by reacting 950 mL of 7 mM ABTS solution with 50 mL of 2.5 mM potassium persulfate, previously left in the darkness at 4 °C for 12 to 16 h. The 50 μL of samples reacted with 250 μL of the ABTS+· solution for 30 min in the dark, and then the absorbance was measured at 735 nm microplate spectrophotometer. The following equation was used to express the ABTS radical-scavenging activity as a percentage (Equation 4):

$$\begin{array}{l}ABTSradicalscavengingactivity\left(\%\right)=\\ \left(1-sampleabsorbance/controlabsorbance\right)\times 100\end{array}$$ (4)

2.7 Cell culture

SH-SY5Y human neuroblastoma cells and Raw 264.7 mouse macrophage cells were purchased from the Korea Cell Line Bank (Seoul, Korea). SH-SY5Y cells were refined in RPMI-1640 medium (Gibco, Waltham, MA, USA) with the addition of 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). Raw 264.7 cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere. Experiment was performed with a 70-80% cell confluency.

2.8 Cell viability and sample treatment

Cells were seeded in a 96-well plate for 24 h and samples were pretreated for 12 h at various naringin (1-50 μg/mL) or yuzu extract concentrations (10-500 μg/mL). The frozen material of each yuzu varieties was homogenized, extracted with 80% ethanol by the soxhlet extraction (60 °C for 3 h) and evaporated to dryness under vacuum for the study. As the stimulant, 5 mM of scopolamine was added to the SH-SY5Y cells and 500 μg/mL of lipopolysaccharide (LPS, E. coli serotype 055:B5) was added to the Raw 264. 7 cells for 24 h. The survival rate was compared and examined, with the percentage representing the difference between absorbance and control. For sample treatment, cells were treated with three varieties of yuzu extracts (10-100 µg/mL) or naringin (5-20 µg/mL) for 24 h.

2.9 Acetylcholinesterase Enzyme-Linked Immunosorbent Assay (ELISA)

The amount of acetylcholinesterase (AChE) was quantified using an AChE ELISA kit (CUSABIO, Wuhan, China). The supernatant of naringin (10 µg/mL) or theanine (10 µg/mL) as positive control, and the yuzu extracts (50-100 µg/mL) treated SH-SY5Y cells were put into each well, incubated at 37 °C for 2 h and then incubated with 100 µL of biotin-antibody at 37 °C for 1 h. Horse radish peroxidase-avidin was added and incubated at 37 °C for 1 h after being washed with wash buffer. Subsequently, 3,3`,5,5`-tetramethylbenzidine substrate was added to the washed well, and after 30 min of 37 °C incubation, the stop solution was added and absorbance was measured at 450 nm.

2.10 Western blot

The SH-SY5Y cells were treated by scopolamine (5 mM) before application of naringin (10 µg/mL) or yuzu extracts (50-100 µg/mL), and then cells were lysed and prepared 30 µg of proteins in each sample. Samples were put together with the molecular weight marker on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was running with the voltage equal to 100 V for 1 h. Next, the whole protein was transferred to the polyvinylidene fluoride membrane to 100 V for 2 h. The membrane was blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Membranes were then incubated overnight at 4 °C with rabbit polyclonal anti-Bcl-2, rabbit polyclonal anti-Bax, rabbit monoclonal anti-PARP, rabbit monoclonal anti-caspase-3 or rabbit polyclonal anti-ß actin antibodies (1:1000, Cell Signaling Technology, Beverly, MA). After repeating washing 4 times with TBST, the secondary antibody was applied for 1 h and the washing process was repeated. Then, 1 mL of western HRP substrate was added and the membrane was developed by ChemiDoc XRS image analysis software. Band density was determined and expression levels were quantified relative to that of β-actin.

2.11 Nitric Oxide (NO) measurement

NO content was measured in the culture medium using the Griess Reagent System (PROMEGA, Madison, WI, USA). The supernatant of sample treated Raw 264.7 cell and nitrite standard (0-100μM) were distributed into each well and 50μL of sulfanilamide solution was treated. After 10 min incubation at 25 °C, 50μL of N-(1-Naphtyl)- ethylenediamine solution was added to each well and absorbance was measured at 540 nm in the 96-well plate.

2.12 Statistical analysis

All values are expressed as mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA). The mean comparison was made by Duncan’s multiple-range test and significant differences were determined at p < 0.05. Statistical analysis were performed using the SPSS 23.0 for Windows software (SPSS Inc., Chicago, IL, USA).

3 Results and discussion

3.1 Physicochemical properties

The appearance of matured yuzu varieties in Native, Tadanishiki and Namhae harvested in November are shown in Figure 1.

Figure 1
Three Korean major yuzu varieties, Native, Tadanishiki and Namhae yuzu in November.

Physicochemical properties of three yuzu varieties like total fruit weight, Hunter color, acidity and soluble solid content are shown in Table 1. Tadanishiki yuzu exhibited the lowest fruit weight (70 g), Native (132 g) but Namhae had the highest fruit weight (141 g).

Table 1
Changes in physicochemical characteristics of yuzu according to yuzu varieties.

	Weight (g)	Hunter's color value			Total acidity	Soluble solids (°Brix)
		L	a	b
Native	132 ± 11a(1)	66.08 ± 1.80b	4.66 ± 0.44ns(2)	42.21 ± 1.08ns	4.66 ± 0.44ns	8.71 ± 0.80ns
Tadanishiki	70 ± 6b	69.14 ± 1.18a	4.48 ± 0.34	43.23 ± 0.95	4.48 ± 0.34	8.81 ± 0.41
Namhae	141 ± 20a	66.05 ± 0.62b	4.48 ± 0.65	41.55 ± 0.51	4.48 ± 0.65	10.11 ± 0.91

• ⁽¹⁾By using Duncan's multiple range test, the mean with the same letter in each column is not significantly different ( p < 0.05). The value represents the mean ± SD (n = 3).⁽²⁾“ns” means not significant.

Hunter value evaluation of yuzu peels showed that the Tadanishiki achieved the maximum L (lightness) and b (yellow) values of 69.14 and 43.23, respectively. Native showed the highest a (redness) value of 4.66 in maturity stages. Native was characterized by the highest acidity and Namhae had the highest soluble solid (°Brix) content among three yuzu varieties.

3.2 Dietary fiber composition

The three yuzu types' TDF content was raised in the following order: Namhae > Tadanishiki > Native (Table 2). The highest contents of IDF and SDF were found in Native (16.12%) and Namhae (21.96%) correspondingly. Based on dietary fiber composition, it was found that the content of this component was increased in the following order: pectin > lignin > hemicellulose > cellulose. Tadanishiki had the lowest lignin (55.53 mg/g DW) and hemicellulose (53.93 mg/g) contents with a value of about 50% compared to native or Namhae. Tadanishiki yuzu without seeds was thought to contain less lignin, which is a main ingredient of the seeds. According to the Birch & Parker (1983) study, an increase in the lignin content was noted with the progress of seed germination, which confirms the results of this study.

Table 2
Dietary fiber contents according to yuzu varieties.

	TDF(1)	IDF(2)	SDF(3)	Lignin	Cellulose	Hemicellulose	Pectin
	(g/100 g DW)			(mg/g DW)
Native	35.51 ± 1.86ns(4)	16.12 ± 2.23ns	19.39 ± 4.09ns	90.53 ± 11.17b(5)	23.27 ± 1.69ns	97.00 ± 6.80b	142.27±17.15b
Tadanishiki	36.30 ± 4.64	15.73 ± 4.21	20.57 ± 0.43	55.53 ± 10.75c	28.83 ± 4.57	53.93 ± 6.21c	176.93±0.65a
Namhae	37.29 ± 3.03	15.33 ± 0.81	21.96 ± 3.84	117.27 ± 7.76a	26.33 ± 4.31	114.37 ± 6.65a	171.00±14.70a

• ⁽¹⁾Total dietary fiber.
• ⁽²⁾Insoluble dietary fiber.
• ⁽³⁾Soluble dietary fiber.
• ⁽⁴⁾“ns” means not significant.
• ⁽⁵⁾By using Duncan's multiple range test, the mean with the same letter in each column is not significantly different by Duncan’s multiple range test (p < 0.05). The value represents the mean ± SD (n = 3).

The high contents of cellulose (28.83 mg/g DW) and pectin (176.93 mg/g DW) were found in Tadanishiki compared to the other two varieties. Therefore, Namhae had the highest dietary fiber content of the three varieties. Dietary fiber is helpful in controlling bowel movements, cholesterol level, lowering postpartum blood sugar level and is considered important enough to be compared to nutrients as it can prevent and treat diseases such as colon cancer, diabetes, constipation and hypertension (Amundsen et al., 2003; Rasane et al., 2015).

3.3 Functional phenolics by HPLC-DAD

Yuzu samples' functional phenolic components and flavonoid content as determined by HPLC analysis (Table 3). Tadanishiki has a greater total phenolic content (367 mg/100 g) than Native and Namhae Yuzu (297 mg/100 g). Similarly, Tadanishiki had the highest flavonoid content at 911 mg, followed by 762 mg in Namhae and 615 mg in Native per g dried sample. Kubola & Siriamornpun (2011) reported that the flavonoid content was more than three times higher in the peel part than in pulp or seeds, and it is believed that Tadanishiki has a higher flavonoid content due to its thicker peel than other varieties. The contents of four types of flavonoids were measured by HPLC, including naringin, narirutin, hesperidin, and neohesperidin as functional components of yuzu. Among flavonoids, the narirutin contained the most, ranging from 228.3 to 352 mg/100 g DW. Within the three varieties, Tadanishiki contained the highest amount of narirutin (352 mg/100 g), hesperidin (261 mg/100 g) and neohesperidin (108.7 mg/100 g) respectively. The content of naringin in the tested yuzu varieties was decreased according to the given order: Namhae (105.8 mg) > Tadanishiki (96.9 mg) > Native (72.5 mg). Previous studies (Moon et al., 2015; Lee et al., 2017) have shown that the main flavonoid yuzu components are hesperidin and naringin, with the hesperidin content higher than that of naringin, which is consistent with this study.

Table 3
Functional compounds, flavonoid contents of yuzu determined by HPLC analysis according to yuzu varieties.

	Total phenolics (mg/100 g)	Total flavonoids (mg/100 g)	DPPH (%)	ABTS (%)	Functional compounds (mg/100g DW)
					Naringin	Narirutin	Hesperidin	Neohesperidin	Total
Native	297 ± 5.00b(1)	615 ± 7.00c	25.63 ± 0.40a	54.04 ± 0.45a	72.50 ± 4.50c	228.30 ± 15.60c	156.90 ± 4.20c	79.10 ± 2.60c	536.80±26.9
Tadanishiki	367 ± 4.00a	911 ± 51.0a	26.72 ± 0.77a	55.81 ± 0.10a	96.90 ± 3.00b	352.00 ± 6.50a	261.00 ± 1.70a	108.70 ± 1.00a	818.60±12.2
Namhae	297 ± 5.00b	762 ± 11.0b	24.28 ± 0.40b	46.23 ± 1.47b	105.80 ± 1.20a	308.40 ± 3.20b	187.80 ± 1.20b	88.20 ± 3.30b	690.20±8.90

• ⁽¹⁾The Duncan's multiple range test finds no statistically significant difference between means with the same letter in each column (p < 0.05). The value represents the mean ± SD (n = 3).

3.4 DPPH and ABTS radical scavenging activity

The DPPH radical scavenging activities of yuzu varieties ranged from 24.28% to 26.72%, with the lowest value of 24.28% at Namhae variety (Table 3). There is a high correlation between the total phenol content and free radical scavenging activity (Byun et al., 2021). This is consistent with the research results stating that the DPPH radical scavenging activity is an indicator of antioxidant activity of phenolic substances (Oki et al., 2002). ABTS radical scavenging activity varied in the range of 46.23-55.81%, with the highest content of 55.8% in Tadanishiki yuzu, and there was no significant difference between the two varieties. For three yuzu varieties, antioxidant capacities determined by the two methods showed good agreement.

3.5 Cell protection effects of different yuzu varieties on SH-SY5Y cells

Here, SH-SY5Y cells were chosen as the study's common model to examine the pharmacological and toxicological potential of AChE inhibitors or in vitro neuronal activity (Baral et al., 2015). Scopolamine was treated to induce cell damage, AChE inhibition, oxidant stress or inflammation (Baral et al., 2015). MTT assay was performed to measure the cell viability of SH-SY5Y by various sample concentrations, as indicated in Figure 2A. The experiment showed that the cell viability decreased significantly at the concentration of 50μg/mL for naringin and 500μg/mL for Native, Tadanishiki, and Namhae (Figure 2A). Therefore, naringin and three yuzu varieties were used at concentration of 20μg/mL and 100μg/mL or less, respectively (Figure 2A). The experiment was conducted to measure cytoprotective effect, AChE inhibition, and oxidative-stress defense proteins expression of the yuzu samples on scopolamine induced SH-SY5Y neuroblastoma cells. The cell viability was decreased by 53% in the scopolamine treated NC group compared to the CON group (100%) (Figure 2B).

Figure 2
Cell protective effects in SH-SY5Y nerve cell according to yuzu varieties. (A) Cell viability; (B) Cell viability (scopolamine treatment); (C) AChE ELISA Assay; (D) The representative images of Bcl-2, Bax, caspase 3 and PARP analyzed by western blotting; (E) Bcl-2/Bax, caspase-3 and PARP expression levels quantified by Western blotting analysis. Theanine or naringin was treated with 10 µg/mL concentration as positive control. Data are shown as means ± SD values of duplicate determinations from three experiments. *p < 0.05, **p < 0.01, ***p < 0.001, vs. compared to the control group, #p < 0.05, ##p < 0.01 and ###p < 0.001, vs. compared to the NC group. According to Duncan's multiple range test, different letters above the bar represent a significant difference between groups (p < 0.05).

Tadanishiki exhibited significantly a dose-dependent increase at 10-100μg/mL concentrations among samples (Figure 2B). In addition, the AChE content was increased about 1.87 times (116 mU) in the negative control (NC) by 5 mM scopolamin treatrment compared to the control (CON) (62 mU) (Figure 2C). All AChE content was significantly lowered in all yuzu samples (98 ~ 76 mU), which is the most low at Tadanishiki (76 mU) at 100μg/mL concentration. This AChE value was similar even to that in positive control, theanine or naringin with 70 or 75 mU, respectively (Figure 2C). Therefore, Tadanishiki was considered to have a superior cytoprotective effect on nerve cells compared to Native or Namhae.

Bcl-2 protein is a apoptosis regulator, controlling of mitochondrial membrane permeability in neurons and lymphocytes (Burlacu, 2003). Bax, an apoptosis-inducing protein of the Bcl-2 family, normally promotes apoptosis-related signals (Lalier et al., 2007). Therefore, the regulation of the expression level of Bcl-2 and Bax proteins was considered to have a significant impact on the inhibition of apoptosis in neurons. Caspase 3 is a major transcription factor and its expression pattern can be used as a biomarker to check indirect degree of damage caused by neurotoxin (Jin et al., 2001). Also, PARP initiates apoptosis when cleaved by caspase 3, being known as a contributing factor in inducing apoptosis (Boulares et al., 1999). The western blotting was used to measure the expression level of Bcl-2/Bax, caspase 3 and PARP proteins and expressed as relative protein content after normalization to ß- actin protein.

Western blot analysis was used to identify key signature factors of activated signaling pathways in order to determine the pathway implicated in the predicament of SH-SY5Y cells. Cell apoptosis modulators, Bcl-2/Bax, caspase-3 and PARP expression levels were quantified by western blotting analysis. Naringin was treated as positive control (Figure 2D-2E). Bcl-2/Bax, caspase-3 and PARP expression were increased by 3-fold, 1.2-old, and 1.4-old in neuroblastoma SH-SY5Y cell (NC group), respectively (Figure 2D-2E), which is in accordance with findings from a previous study (Puangmalai et al., 2017; Maugeri et al., 2021). However, in naringin group, Bcl-2/Bax, caspase-3 and PARP expression were almost decreased by 50%, compared to that of NC group (Figure 2D-2E). Among cell apoptosis modulators, Bcl-2/Bax protein expression was significantly diminished by Tadanishki and Namhae groups (0.7 and 0.9 ratio) than by native group (1.5 ratio), and this was lower than naringin value (1.2 ratio). Caspase-3 and PARP showed also slightly reduced protein expression by all yuzu varieties but no significance within samples (Figure 2D-2E).

3.6 Anti-inflammatory effects of yuzu variety on Raw 264.7 cells

An experiment was carried out utilizing Raw 264.7 macrophage cells to verify the anti-inflammatory effects of yuzu varieties, as shown in Figure 3. Since LPS at 500 ng/mL concentration was used as an inflammation inducer to macrophages (Rhule et al., 2006). We measured the macrophage cells cytotoxicity of naringin and yuzu samples for 50μg/mL and 500μg/mL or dosages, respectively. Naringin and yuzu varieties showed relatively low cytotoxicity with 90% cell viability at 100 µg/mL and 10 µg/mL or less, respectively (data not shown). Before LPS application, Raw 264.7 cells were treated with the yuzu samples (50 or 100 µg/mL), or naringin (1 or 10 µg/mL). The cell viability was decreased by 55% in the NC group compared to the CON group (100%), but significantly increased up to 64% ~ 76% in all yuzu samples (Figure 3A). In addition, LPS-induced inflammation resulted in NO production by iNOS expression. When such NO is overproduced, it promotes the generation of numerous free redicals in the body, leading to chronic inflammatory diseases (Dat et al., 2012). In the LPS-treated group, the amount of NO production increased about 26 times (10.4 µM) compared to the CON group (0.4 µM), but it significantly decreased in all yuzu samples (7.1 ~ 8.9 µM), which is similar NO content in 1 μg/mL of naringin (7.8 µM) (Figure 3B). This pattern agreed with a previous reports concerning NO content in LPS-induced RAW 264.7 cells by Citrus unshiu extract, with 15.4% or 40% lower NO production (Seo et al., 2015; Kim et al., 2019).

Figure 3
Anti-inflammatory effects in Raw 264.7 cells according to yuzu varieties. (A) Cell viability; (B) Nitric Oxide (NO) contents. Data are presented as means ± SD values of duplicate determinations from 3 separate experiments. ***p < 0.001, vs. compared to the control group, #p < 0.05, #p < 0.01 and ###p < 0.001, vs. compared to the NC group.

Tadanishiki and Namhae showed a significant effect compared to the Native in both higher cell viability and lower NO production results. Therefore, Tadanishiki and Namhae were considered to be more effective samples for inhibiting of inflammation compared to Native.

4 Conclusion

Natural compounds and their purported role in the prevention of various diseases have aroused people's curiosity in recent years. In this study, Tadanishiki variety of yuzu showed a promising neuroprotective effect which is much higher than in the Native variety. In conclusion, the results from this study suggest that Tadanishiki may be beneficial yuzu varieties for the protection of brain nerve cells.

Acknowledgements

This study was financially supported by the Agriculture Science and Technology Development program (PJ016161) of Rural Development Administration by Korea government.

• Practical Application: These data could offer valuable information for the practical use of major yuzu varieties, specially Tadanishiki yuzu with the highest hesperidin and neuroprotective function. Since Tadanishiki variety with no seed, could be more applicable in food industry with enriched citrus juice and high economic value.

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Publication Dates

• Publication in this collection
  19 Aug 2022
• Date of issue
  2022

History

• Received
  28 May 2022
• Accepted
  19 July 2022