Microbial Biotechnology
Microbiol. Biotechnol. Lett. 2020; 48(4): 471-479
https://doi.org/10.48022/mbl.2004.04008
Nari Lee, Su Bin Hyun, Suk Hyun Yun, You Chul Chung and Chang-Gu Hyun*
Department of Chemistry and Cosmetics, Jeju National University, Jeju 63243, Republic of Korea
Correspondence to :
Chang-Gu Hyun, cghyun@jejunu.ac.kr
The aim of this study was to investigate the anti-oxidant and anti-inflammatory activities of the Rhododendron weyrichii flower extract fermented using Shindari, a traditional Jeju barley Nuruk-based fermentation. In this study, we examined the antioxidant potential of R. weyrichii flower extracts (RF) and R. weyrichii flower extracts fermented with Nuruk or Shindari (RFFN or RFFS, respectively) using various in vitro antioxidant assays including DPPH and ABTS radical scavenging assays, total phenol content and FRAP assays. We also evaluated the anti-inflammatory activity of the RF and RFFS on murine RAW 264.7 cells. The anti-inflammatory activity was evaluated by treating the RAW 264.7 cells with various concentrations (6.25, 12.5, 25, and 50 μg/ml) of RF or RFFS. As a result, we observed that the ABTS radical scavenging activity and total phenol content of RFFS was higher than that of RF and RFFN. Additionally, lipopolysaccharide- induced nitric oxide (NO) production was significantly lower in RFFS-treated cells when compared to the LPS-treated control. In addition, RFFS-treated cells exhibited decreased expression of inducible NO synthase (iNOS) proteins and high-performance liquid chromatography (HPLC) fingerprinting showed that both the quercetin and quercetin glucoside (quercitrin and isoquercitrin) levels were affected by the fermentation process. In conclusion, our data suggests that traditional fermentation could be an important strategy in improving the biological properties of raw materials including their antioxidant and antiinflammatory activities. Finally, RFFS may be a candidate for developing topical antioxidant and antiinflammatory agents.
Keywords: Fermentation, Rhododendron, Nuruk, inflammatory, Shindari
Nuruk is the Korean traditional fermentation starter. The shape, manufacturing process, and fermentation period of Nuruk varies depending on the unique climatic condition of each region [1−3]. In Jeju, red barley is used for the preparation of traditional Nuruk, because, Jeju province has very few rice paddies, Nuruk is prepared from barley rather than rice. Jeju barley Nuruk is also used for brewing a traditional fermented drink called Shindari. It is made by fermenting barley Nuruk and steamed rice for a short period of time [4].
The process of fermentation has been reported to improve the biological properties of the raw material, including the antioxidant, anti-inflammatory, and skinwhitening properties by the production of new bioactive compounds [5−7]. Particularly, Nuruk contains various microorganisms that are involved in saccharification and alcohol fermentation. Therefore, these microorganisms have the potential to improve the biological properties of raw materials.
The lipopolysaccharide (LPS) from
We procured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ammonium pyrrolidinedithiocarbamate (APDTC), protease inhibitor cocktail from Merck (Germany). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/ streptomycin, trypsin-ethylenediaminetetraacetic acid, BCA protein assay kit were purchased from Thermo Fisher Scientific (USA). Dimethyl sulfoxide (DMSO), radioimmunoprecipitation (RIPA) buffer, and enhanced chemiluminescence (ECL) kit were purchased from Biosesang (Korea) and Laemmli sample buffer (2X) was purchased from Bio-Rad (USA).
Subsequently, the fermented extract was prepared in two ways. First, the dried RF extract (1 g) and Nuruk (barley yeast, 1 g) was mixed with 1 g of sugar and added to 50 ml distilled water. Another one was mixed with 1 g of dried RF extract (1 g) and Nuruk (barley yeast, 1 g) with steamed rice and added to 50 ml distilled water. It was prepared by reference to Shindari methods. And then each mixture was fermented for 3 days in an incubator at 30℃.
After fermentation is over, 117 ml of 95% ethanol was added to each fermented mixture to prepare a 70% ethanol extract and stirred at room temperature for 24 h. The extracts were filtered and concentrated by a rotary evaporator. And then, freeze-dried for use in the experiment. The yield of the
The antioxidant activity of the extracts was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay following the protocols of Blois
Radical scavenging activity (%) = [1 – ((Asample - Ablank) / Acontrol)] × 100
The ABTS+ radical scavenging activity was measured following the methods of Re
The total polyphenol content was determined by colorimetric assay following the method described by Folin-Denis (1981) [13]. Briefly, the extract (100 μl) was diluted to a total volume of 1 ml using 900 μl distilled water. The extract was incubated with 100 μl Folin- Ciocalteu’s phenol reagent at room temperature for about 3 min. Further, 200 μl of Na2CO3 solution (7%, w/v) was added and mixed, followed by the addition of 700 μl distilled water. This mixture was incubated at room temperature for 1 h. The absorbance of the reaction mixture was measured at 700 nm using a microplate reader. The total polyphenol content was quantified using the gallic acid standard curve (R2 = 1).
The ferric reducing capacity of the extracts was evaluated following the method described by Benzie and Strain (1996) [14]. Briefly, the FRAP reagent was prepared using 300 mM sodium acetate buffer (pH 3.6), 10 mM 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ, dissolved in 40 mM HCl), and 20 mM ferric chloride solution in the ratio of 10:1:1. The extract (20 μl) was incubated with 180 μl of the FRAP reagent at 37℃ for 10 min. The absorbance of the reaction mixture was measured at 590 nm using a microplate reader. The ferrous (Fe2+) ion was quantified using the FeSO4·7H2O standard calibration curve (R2 = 0.9996).
The mouse monocyte cell line, RAW 264.7 was obtained from the Korean Cell Line Bank (KCLB). The cells were sub-cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin every second day and maintained in a humid atmosphere of 5% CO2 at 37℃.
The cell viability was measured using 3-(4,5-dimethylthiazol- 2yl)-,5-diphenyltetra-zolium bromide (MTT) assay [15]. The RAW 264.7 cells (1 × 105 cells/well) were seeded in the 24-well plates. Further, the cells were treated with various concentrations of the extract for 24 h. The MTT solution was added to each well. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at 570 nm using a microplate reader (SUNRISE, TECAN Austria GmbH).
The RAW 264.7 cells (1 × 105 cells/well) were seeded in the 24-well plates. The cells were then co-treated with RF or RFFS (6.25, 12.5, 25, and 50 μg/ml) and lipopolysaccharide (LPS: 1 μg/ml) for 24 h. The culture supernatant was incubated with an equal volume of griess reagent at room temperature (22−25℃) for 10 min. The NO production was measured at 540 nm using a microplate reader.
The RAW 264.7 cells were seeded in the 60-mm dish (2.5 × 105 cells/dish) and cultured for 24 h. The cells were treated with various concentrations of RFFS (12.5, 25, and 50 μg /ml) or APDTC (15 μM) and LPS (1 μg/ml) for 24 h. The cells were subjected to trypsin treatment and lysed by vortexing every 10 min for 1 h in RIPA buffer containing 1% protease inhibitor cocktail. The cells were centrifuged at 21055 ×
The quantitative analysis of the RF, RFFN, and RFFS extracts was performed on an HPLC instrument with a 2695 separation module (Waters, USA). The column used for the separation of RF, RFFN, and RFFS extracts was the YMC-Triart C18 analytical column (250 mm × 4.6 mm, 5 μm, 12 nm, Waters 2695), which was maintained at 30℃. The mobile phase used for the efficient separation of the analytes was acetonitrile and 20 mM phosphoric acid. The flow rate was constantly maintained at 1.0 ml/min and the injection volume was 10 μl. The analytes were detected using the PDA detector. The wavelength range used for the quantification of analytes was 280−370 nm. The data analysis was performed using the Waters Empower software. The RF, RFFN, and RFFS extracts were dissolved in 70% ethanol at a concentration of 10 mg/ml.
Cell viability and NO production assay were repeated at least four times, and each experiment was performed in triplicate. All data are presented as mean ± standard deviation (SD). *
Evaluation of antioxidant activity of RF, RFFN, and RFFS The antioxidant activity of the phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. The FRAP method is based on the reduction of ferric-TPTZ complex to ferrous-TPTZ in the presence of antioxidants [16−18].
The total phenolic content, FRAP value, and radical scavenging activity of RF, RFFN and RFFS are presented in Table 1. We have observed that RF, RFFN, and RFFS have high antioxidant efficacy. The FRAP value of RFFS was 1.4-fold higher than that of RFFN. Further, we observed that the phenolic content of RFFS was 1.1-fold, and 2.1-fold higher than that of RF, and RFFN. The IC50 value of RFFN and RFFS for ABTS scavenging activity was lower than that of the positive control, ascorbic acid. The IC50 value of RFFS was 4.88- fold lower than that of ascorbic acid and 1.4-fold lower than RF. Additionally, the radical scavenging activity of RFFN and RFFS was dose-dependent (Fig. 1). However, as can be seen from the experimental results, the fermentation process of
Table 1 . Antioxidant effect of RF, RFFN, and RFFS.
Sample | 1)IC50 value (μg/ml) | Total phenolic content (mg 5)GAE/) | FRAP (mM Fe2+/g) | |
---|---|---|---|---|
DPPH radical scavenging activity | ABTS radical scavenging activity | |||
Ascorbic acid | 7.33 ± 0.26 | 24.50 ± 0.59 | - | - |
2)RF | 21.896 ± 0.31 | 7.363 ± 0.23 | 407.494 ± 2.55 | 2.767 ± 0.12 |
3)RFFN | 35.98 ± 0.10*** | 12.49 ± 0.32*** | 217.073 ± 24.06*** | 0.839 ± 0.02*** |
4)RFFS | 24.51 ± 0.28** | 5.02 ± 0.12** | 455.695 ± 23.95* | 1.132 ± 0.03*** |
1)IC50 : Concentration required to inhibit 50% of radicals
2)RF :
3)RFFN :
4)RFFS :
5)Gallic acid equivalent
The data are expressed as mean ± SD (n = 3). **
In this study, we investigated the effect of
Western blotting was performed to determine whether NO production was associated with the protein expression of iNOS. The expression of iNOS in the LPS-induced RAW 264.7 cells was significantly higher than that in the LPS-untreated cells. The co-treatment of RFFS (50 μg/ml) and LPS significantly reduced the expression of iNOS by 61% compared to the LPS-treated group. The negative control, APDTC inhibited the expression of iNOS by 80% at 15 μM compared to the LPS-treated group (Fig. 4). This indicated that RFFS suppresses the LPS-induced inflammation in the RAW 264.7 cells by inhibiting the NO production and iNOS expression.
HPLC fingerprinting was used to identify the chemical components of
Table 2 . Chemical constituents of RF, RFFN, and RFFS. The concentration of all standard materials was measured at 100 ppm.
Sample | Quercitrin | Isoquercitrin | Quercetin (ppm) |
---|---|---|---|
STD | 100 | 100 | 100 |
1)RF | 23.74 | 19.73 | 19.73 |
2)RFFN | 17.07 | 8.54 | 51.16 |
3)RFFS | - | - | 83.50 |
1)RF:
2)RFFN:
3)RFFS:
Nuruk (yeast) is a traditional fermented starter that is manufactured in a wide variety of forms and recipes, depending on the geographic environment of the region. Jeju Island, a volcanic island, has been used for traditional fermented foods by using barley to make Nuruk. In particular, Shindari is a traditional drink that is fermented for 3 to 4 days in a short time by adding barley Nuruk to the steamed rice [23]. Various beneficial microorganisms are present in these Nuruk, and vitamins, essential amino acids, and organic acids produced through the fermentation process affect nutrition and flavor [24]. In addition, previous studies have been conducted on the effects of Nuruk in various aspects such as antioxidants and whitening [25, 26].
In this study, we demonstrated the antioxidant potential of
HPLC fingerprinting suggest that deglycosylation by microorganisms during Shindari fermentation may degrade glycosides of quercitrin and isoquercitrin to increase the content of quercetin, which is known to exhibit antioxidant and anti-inflammation activities.
Hence, Shindari fermentation has the potential to improve biological properties such as antioxidant and anti-inflammatory properties. Thus, we suggest that REFS is a potential antioxidant and anti-inflammatory candidate for topical application. In order to use these results, a complete understanding of the efficacy of the fermentation starter, barley Nuruk or Shindari, and the increased efficacy after fermentation of the extract is needed. Therefore, it is considered that it is necessary to further compare the efficacy of the fermentation starter and the fermentation extract under the same conditions.
This work was supported by the Academic and Research Institutions R&D Program (C0516709) funded by the Ministry of SMEs and Startups (MSS, Korea).
The authors have no financial conflicts of interest to declare.
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