*4.8. Determination of Nitrite (NO) and Prostaglandin E<sup>2</sup> (PGE2)*

To determine the inflammation level in RAW 264.7 cells, the Griess assay was used [50]. Griess reagent (1% sulfanilamide/0.1% N-(1-naphtyl)ethylene diamine dihydrochloride in 2.5% H3PO4) was mixed with an equal part of the cell culture medium of control or various experimental groups RAW 264.7 cells. In this test, the NO<sup>2</sup> content was used as an indicator of NO content in RAW364.7 macrophages. The OD 550 nm was determined and calibrated using a standard curve of NaNO<sup>2</sup> prepared in culture medium.

The PGE<sup>2</sup> levels in RAW264.7 macrophages were determined using an enzyme immunoassay (EIA) kit (ADI-900-001, Cayman Chemical, Ann Arbor, MI, USA). The cell culture supernatants were collected after experimental treatment and centrifuged at 1000× *g* for 15 min to remove the particulate matter. The medium and PGE2 EIA conjugate was added to a 96-well plate pre-coated with goat anti-mouse IgG and left to react for 1 h, followed by a final wash to remove any unbound antibody-enzyme reagent. A substrate solution was added and the intensity of the color produced was measured at 412 nm. The concentration of PGE<sup>2</sup> in each sample was calculated according to PGE<sup>2</sup> standards.

### *4.9. Measurement of IL-1β, IL-6 and TNF-α*

The levels of IL-1β, IL-6 and TNF-α in RAW264.7 macrophages were analysed using rat IL-1β/IL-1F2 DuoSet ELISA (R&D, DY501-05), rat IL-6 DuoSet ELISA (R&D, DY506-05), and rat TNF-α DuoSet ELISA (R&D, DY510-05) kits (R&D Systems, Inc., Minneapolis, MN, USA), respectively, according to the manufacturer's instructions. In brief, capture antibodies, cultured medium supernatants, detection antibodies, streptavidin-conjugated horseradish-peroxidase were processed on the plate in order, and the color subtract tetramethylbenzidine was used. The absorbance was measured and the concentration was calculated according to the standard.

### *4.10. Immunoblot Analyses of iNOS, COX-2 and NF-κB Signalling Molecule Expression*

The iNOS, COX-2 and NF-κB signalling molecule expression was analysed using the method described by Hsieh et al. [51]. At the end of the treatment, the cells were collected in 200 µL of lysis buffer (10 mM Tris-HCl, 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride and 20 µg/mL aprotinin, pH 7.4), and the protein content was determined according to the method of Lowry et al. [44].

Equal amounts (approximately 10–20 µg per sample) of cellular protein were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) [52], after which the samples were transferred to polyvinylidene difluoride (PVDF) membranes [53]. The PVDF membranes were then incubated with anti-iNOS, anti-COX-2, anti-p-IκB, anti-IκB, anti-NF-κB (p65) or anti-GAPDH antibodies at 4 ◦C overnight, followed by incubation with a peroxidase-conjugated secondary antibody. For density analysis, blots were treated with enhanced chemiluminescence substrate solutions and exposed using a ChemiDoc XRSt System (Bio-Rad Laboratories, Hercules, CA, USA). An NF-κB (p65) transcription factor activity assay kit (Cayman Chemical Co.) was used to analyse the NF-κB DNA binding activity of the nuclear fraction.

### *4.11. Statistical Analysis*

The SPSS Statistical Analysis Software for Windows, version 20.0 (SPSS Inc., Chicago, IL, USA) was used to analyse the experimental data in the present study. One-way analysis of variance (ANOVA) and Duncan's or Tukey's multiple-range test were used to evaluate the significance of differences between each mean value. A *p*-value less than 0.05 was used to indicate a statistically significant result.

### **5. Conclusions**

In conclusion, the presented data in this study demonstrate that BBSLP could reduce oxidative stress and pro-inflammation factors in LPS-induced RAW264.7 cells through the inhibition of the NF-κB signaling pathway, indicating that it may have potent antioxidant and anti-inflammatory capabilities, suggesting that BBSLP could be developed as a supplement material for functional foods.

**Author Contributions:** Conceptualization, S.-L.H., S.-F.T. and C.-C.W.; Methodology, S.-L.H. and Y.-M.C.; Validation, Y.-W.S., C.-C.L. and C.-C.W.; Formal Analysis, Y.-M.C.; Investigation, Y.-W.S. and S.-L.H.; Resources, Y.-M.C. and S.-F.T.; Data Curation, Y.-W.S. and C.-C.W.; Writing—Original Draft Preparation, C.-C.W.; Writing—Review & Editing, S.-L.H. and C.-C.W.; Supervision, C.-C.W.; Project Administration, C.-C.W. All Authors read and approved the article and agree to be accountable for all aspects of the research and ensure that the accuracy or integrity of any part of the work is appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Ta-Tung Soya Sauce Co. Ltd. through a grant (grant no. PU108-11150-A002).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

