Evaluation of antioxidant and anti-inflammatory effects of three different Rubus coreanus Miq. by-products

Purpose: To investigate the antioxidant and anti-inflammatory properties of three different Rubus coreanus Miq. by-products in stimulated BV-2 microglial cells and explore its underlying physiological efficacy. Methods: Cell viability assessment was performed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay. Lipopolysaccharide (LPS) was used to activate BV-2 microglia. Total polyphenol and total flavonoid contents were determined by the method of Folin-Denis. As three different Rubus coreanus Miq. by-products remaining after extraction of Rubus coreanus, High performance liquid chromatography (HPLC)) finger printing, ABST (2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay, and NO (nitric oxide) inhibitory assay were performed. Results: Three different Rubus coreanus by-product extract did not exhibit any signs of cytotoxicity to BV-2 cells up to 100 μg/ml concentration (p < 0.5). The LPS-activated excessive release of NO in BV-2 cells was significantly inhibited by Rubus coreanus by-product extract (p < 0.5) at 500 μg/mL). Total polyphenol and total flavonoid contents were highest in 50 % ethanol wine processing by-product (p < 0.5 at 30, 50, 70 and 100 %, respectively). The by-product of wine processing had the lowest RC50 radical scavenging effect (16.53 μL/ml). The quercetin content of the wine processing by-product was the highest in the 70% ethanol extract at 6.26 mg/g (p < 0.5 at 30, 50, 70 and 100%, respectively). Conclusion: These results reveal that of the three other by-products, wine processing by-product has the highest antioxidant and anti-inflammatory activities. The use of these by-products has high added value for industrial production; furthermore, they are a potential treatment for various inflammatory diseases.


INTRODUCTION
Rubus coreanus Miq. is a deciduous broadleaf shrub belonging to the family Rosaceae that grows in to the far-east Asian countries such as China, Japan, and South Korea [1]. In Korea, it is called 'bokbunja', and has been used for treating asthma, enuresis, spermatorrhea, and allergic diseases in oriental medicine [2]. In general, among the components of R. coreanus (100 g), moisture 87.0 g, protein 1.6 g, fat 1.7 g, glucose 1.3 g, fiber 2.9 g, ash 0.5 g, etc. are variously contained. As inorganic salts, potassium (K), Calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), and the like, and vitamins contain vitamin A, vitamin B1, B2, and vitamin C [3]. In addition, it is reported to have functions such as anti-cancer activity, antioxidant activity, cholesterol-lowering effect and anti-tumor activity [4] because it contains useful compounds such as gallic acid, tannin, catechin, and quercetin [5].
Although R. coreanus is mainly consumed as a raw fruit, its use is increasing as a raw material for wine and juice due to an increase in preference for natural fruit juice, and recently, it has been used in various ways such as jelly and jam [6]. Rubus c. processing by-products are about 20% of the total R. coreanus, and some are used as feed, but most of them are discarded [7]. Ku et al. [7] reported that the hot water extract of R. coreanus processing by-products contained organic acids 8.9 %, neutral sugar 69.9 %, pectin 19.1 %, anthocyanins 5.2 %. Jeong and Seo [8] reported a study on the polyphenol content of R. coreanus processing by-products, but studies using R. coreanus processing by-products are rare. In the body, biochemical reactions constantly take place to supply the energy required for metabolism, thereby generating free radicals [9]. These free radicals are continuously produced as by-product of metabolic processes in the body, and are unstable and highly reactive [10]. When oxidative stress occurs because excessive active oxygen is generated in the body and cannot be cleared through normal metabolism [11], cells and tissues are damaged, leading to various degenerative diseases such as cancer, heart disease, arteriosclerosis, dermatitis, and dementia [12]. Herbal medicines are known to contain large amounts of antioxidants that can remove active oxygen. The aim of this study is to promote the potential utilization of R. coreanus processing by-products a as medicinal herb. Therefore, in this study, extracts were prepared by different extraction temperatures and ethanol ratio conditions of raw R. coreanus and byproducts generated after processing juice and wine of R. coreanus, and optimal extraction conditions were established through antioxidant and anti-inflammatory screening.

Preparation of R. coreanus extracts
Raw fruits of R. coreanus were collected from Gochang (Jeollabukdo, Korea) on August, 2019. Juice and wine by-products of R. coreanus were obtained from a processing plant of R. coreanus in Gochang (Jeollabukdo, Korea). The various extraction conditions of R. coreanus are shown in Table 1. All extracts were extracted for 24 h.

Total polyphenol and flavonoid contents (TPC and TFC)
Total polyphenol content of R. coreanus extract was determined by the method of Folin-Denis [13]. After the R. coreanus extract was prepared at a concentration of 1 mg/mL, Folin & Ciocalteau's phenol reagent 60 μL was added to sample solution 60 μL. After 3 min, 60 μL of a 10% sodium carbonate (Na2CO3) (Sigma Chemical Co., St. Louis, Mo. USA) was added, allowed to stand at RT for 1 h. The absorbance of mixture was read at 750 nm (Bio-Rad Inc., Hercules, CA, USA). The standard curve was used to calculate the total polyphenol content of the sample extracts which was expressed as gallic acid (Sigma Chemical Co.) equivalent mg (GAE)/g dry weight. Total flavonoid content of R. coreanus extract was evaluated by a colorimetric method described by Nieva Moreno et al. [14]. After diluting the R. coreanus extract (100 μL) was mixed with 20 μL of 1 M potassium acetate and 10% aluminum nitrate adding 860 μL of 80% ethanol. After 40 min, the absorbance of the reaction mixture was then measured at 415 nm (Bio-Rad Inc.). The total flavonoid content was determined with respect to the standard curve of quercetin (Sigma Chemical Co.), and was expressed as quercetin equivalent.

ABTS radical scavenging activity
ABTS radical scavenging activity of R. coreanus extract was determined by the method of Re et al. [15]. ABTSꞏ+ cation radical was produced by the reaction between 7 mM ABTS in PBS and 2.45 mM potassium persulfate (1:1), stored in the dark at room temperature for 24 h before use. ABTSꞏ+ solution was then diluted with PBS to obtain an absorbance of 0.70 ± 0.02 at 734 nm. After the addition of 20 μL of R. coreanus extract to 180 μL of diluted ABTSꞏ+ solution, the absorbance was measured at 1 min after the initial mixing. The ABTS radical scavenging activity was calculated by the following equation. ABTSꞏ+ scavenging effect (%) = ((AB-AA)/ AB)×100, where, AB is absorbance of ABTS radical + methanol; AA is absorbance of ABTS radical + sample extract/standard.

Identification of quercetin by HPLC
The content of quercetin in R. coreanus extract was analyzed by the method of Yang et al [16].
The R. coreanus extract was dissolved in 99 % methanol to prepare solutions at 10 mg/mL. Solutions were filtered through 0.22 μm filters. Using a 20 μL sample loop, the sample was analyzed using a reversed-phase HPLC system (Waters 2690, MA, USA), a quaternary pump, and a vaccum degasser. Quercetin separation was performed by a prepacked ZORBAX C18 column (XDB-C18, 4.6 × 150 mm, 5 μm). Two mobile phases were used: solvent A, 5% acetic acid; B, 100% acetonitrile. The gradient for HPLC analysis was linearly changed as follow (total 50 min): 5 % B at 0 min, 25 % B at 20 min, 100 % B at 21 min, 100 % B at 35 min, 5 % B at 36 min, 5 % B at 50 min. Flow rate was set to 1.0 mL/min.

Cell culture, cytotoxicity, and nitrite measurement
BV-2 microglia cells were cultured in RPMI1640 (Gibco BRL, NY, USA) containing 10% fetal bovine serum (FBS; Gibco BRL) and 1 % antibiotic-antimycotic (Gibco BRL) at 37°C in 5% CO2. Cell viability against R. coreanus extracts its was measured by a 3-[4,5-Dimethylthiazol-2yl] -2,5-diphenyl-tetrazolium bromide (MTT) assay. Cells (1 x 10 5 cell / mL) were dispensed in 100 μL in 96 well-plates and cultured in a CO2 incubator for more than 12 h, and then followed by incubation for 24 h by treating 100 μL of fresh medium and R. coreanus extract (100 and 500 μg/mL). After 24 h, 10 μL of MTT (2.5 mg/mL) was added, followed by incubation for 4 h. And then, the culture medium was removed, and 100 μL of dimethylsulfoxide (DMSO) was added to dissolve the generated formazan crystals, which was measured at 540 nm using a microplate reader. Cell viability was expressed as a percentage (%) compared to the control. The inhibitory effect of nitric oxide (NO) was measured by reacting the NO2form present in the cell culture with Griess Reagent using the method of Green et al [17]. BV-2 cells (1 x 10 5 cell/mL) were cultured in 96-well plates overnight. The cells were treated with R. coreanus extracts (100 and 500 μg/mL) and 100 ng/mL of lipopolysaccharide (LPS) for 24 h. And then, 100 μL of cell culture solution and 100 μL of Griess reagent (Sigma Chemical Co.) were mixed and reacted in 96 well-plates for 10 min, and then measured at 540 nm using a microplate reader.

Data analysis
The results were expressed as mean ± SD and statistically analyzed by analysis of variance (ANOVA). Duncan's multiple range test was performed to determine significant differences among the groups, and differences at ρ < 0.05 were considered significant.

Total polyphenol (TPC) and total flavonoid contents (TFC)
Total phenolic and flavonoid contents of R. coreanus processing by-products extracted by using different concentration of ethanol and different temperature of water are presented in figure 1. The results showed that the recovery of phenolic and flavonoid compounds was significantly varied depending on the temperature of water and ratios of the ethanol used. The TPC of water temperature-based extract ranged from 35.94 mg GAE/g to 105.17 mg GAE/g, from 29.72 mg GAE/g to 242.52 mg GAE/g for the ethanol concentration-based extract, respectively. Besides, all the extracts of wine processing by-product showed the highest amounts of TPC (Fig. 1A).
The results of TFC were expressed as mg quercetin equivalent (QE) per gram dry extract. The TFC of water temperature-based extract ranged from 13.21 mg QE/g to 55.62 mg QE/g, from 10.27 mg QE/g to 72.68 mg QE/g for the ethanol concentration-based extract, respectively (Fig. 1B).  Figure 2 shows RC50 values of R. coreanus processing by-products extracted by using different concentration of ethanol and different temperature of water. It can be seen from the results that the wine processing by-product showed the lowest RC50 value, demonstrating high antioxidant activity, followed by raw R. coreanus and juice processing by-products, respectively.

Figure 2:
ABTS radical scavenging activity of the raw R. coreanus and by-products by various extraction conditions. RC50 is amount required for 50% reduction of ABTS radical scavenging activity. Data are mean±standard deviation (n=3). Different superscripts in a column indicate significant differences at p < 0.05 by Duncan's multiple range test

Quercetin content
In this study, the quercetin contents of raw R. coreanus and by-products generated after processing juice and wine of R. coreanus by various extraction conditions was analyzed by HPLC analysis, and presented in figure 3. The content of quercetin was in the range of 0 -6.26 mg/g, and the extracts of wine processing byproduct was contained the highest quercetin content. Overall, the extraction method showed optimal quercetin extraction when 50% ethanol solvent was used.

Cytotoxicity
To determine the cell cytotoxicity of R. coreanus processing by-products extract treatments, we carried out the MTT assay to measure the viability of cells cotreated with R. coreanus processing by-products extract/LPS (Figure 4). Based on our results, treatments of raw R. coreanus and juice processing by-products up to 500 μg/mL for 24 h had no cytotoxic effects in comparison with the control group. However, 70 and 100 % ethanol extract of wine processing byproduct at a dose of 500 μg/mL significantly reduced the viability of BV-2 cells with LPS.

Nitric oxide (NO) inhibitory effects
We examined the anti-inflammatory effect of R. coreanus processing by-products extract by investigating the levels of the proinflammatory mediators NO in LPS-stimulated BV-2 cells (Figure 1.B). Our results confirmed the water extract had no NO inhibition effect, but the ethanol extract showed a NO inhibition effect ranging from 14.94 to 67.72% excluding cytotoxic concentrations. Especially, 50% ethanol extract of wine processing by-product have been proven as the best extraction condition for NO inhibition effect.

DISCUSSION
R. coreanus is well known to have various pharmacological effects, including antiinflammatory and antioxidant [2] activities, which are attributed to the phenolic compounds such as anthocyanin, gallic, vanillic, caffeic acids, phydroxybenzoic, salicylic, syringic, and protocatechuic. [3,5]. However, the effect of R. coreanus processing by-products and extraction conditions on the antioxidant and antiinflammatory effects of R. coreanus have not yet been examined. This is the first study to compare the antioxidant and anti-inflammatory effects of different extraction condition extracts of R. coreanus processing by-products.
Phenolic compounds, which are widely distributed in medicinal herbs, are well known as natural antioxidants that act as reducing agents, hydrogen or electron donors, metal cations chelators, and singlet oxygen quenchers [18]. Polyphenol compounds have an aromatic ring with two or more phenolic hydroxyl groups in the molecule, and there are more than 1,000 types such as phenolic acid derivatives, anthocyanins, flavonoids, lignans, resveratrol and tannins [19].  These results clearly showed that the order of the extracts with the highest level of TPC in wine processing by-product was as follows: 50 % ethanol > 30 % ethanol > 100 % ethanol > 70% ethanol > 80 o C water > 40 o C water > 25 o C water extract. Among the kind of processing byproduct, wine processing by-product extracted using 50% ethanol showed the highest amount of TFC. Similar to TPC, TPC and TFC of wine processing by-product were the highest among the processing by-product irrespective of the extraction condition used. Previously, many researchers reported that the increase in total phenolic during fermentation of natural materials was a result of enzymatic reactions such as microbial-derived β-glucosidase or esterase [20]. In this study, the increase in TPC and TFC in wine processing by-products is thought to be a result of several enzymes generated from the metabolism of yeast used in wine processing.
In the present study, ABTS free radicalscavenging assays was used to determine the antioxidant capacity of different extraction condition extracts of R. coreanus processing byproducts. The antioxidant activity is usually expressed either as percentage reduction of ABTS or RC50, which indicates 50% inhibition of ABTS radicals. The lowest RC50 value shows the most potent antioxidant activity. In the present work, the scavenging activity was expressed as RC50 (μg/mL). Wine processing by-product showed the lowest RC50 value, demonstrating high antioxidant activity. This result was correlated with the highest TPC and TFC in the 50% ethanol concentration of wine processing by-product. 50 % ethanol extract of wine processing by-product have been proven as the best extraction condition for the extraction of antioxidants.
Quercetin is a flavonoid with antioxidant and antiinflammatory properties [21]. This compound is present in fruit, vegetables, and is abundant in red wine [22]. In this study, it was confirmed that quercetin extract, a bioactive compound, was the highest when extracted with 50% ethanol from wine processing by-product of R. coreanus Song et al. [23] showed optimal extraction results in the 40 -80% ethanol ratio of the extraction efficiency of polyphenols, flavonoids, and chlorogenic acid, which are bioactive compounds of Lonicera japonica Flos. Therefore, the ratio of the extraction method and solvent should be considered according to the bioactive compound to be extracted.
Among the inflammatory mediators, reactive oxygen species, nitric oxide (NO) in particular, initiates a wide range of toxic oxidative reactions causing tissue injury [24]. Our results clearly showed that R. coreanus ethanol extract attenuated NO production. Similar to the antioxidant results, 50% ethanol extract of wine processing by-product was the most attenuated NO production without cytotoxicity. This result indicates that 50 % ethanol extract of wine processing by-product acts principally by regulating NO generation and could be beneficial for preventing the progression of neuroinflammation by BV-2 microglial activation.

CONCLUSION
The findings of this study show that wine processing by-product of R. coreanus inhibits inflammatory responses in LPS-stimulated BV-2 microglial cells and has the potential to be developed into a therapeutic agent for the treatment of various inflammatory diseases.