Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells
Okra is the well-known vegetable, green pods, within the Asian cuisine. This study aimed to evaluate the antioxidant activities of okra seed protein hydrolysate produced by combining three enzymes (trypsin, chymotrypsin, and pepsin). The hydrolysate was filtered through an ultrafiltration membrane w...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
|
| Series: | Applied Food Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772502225002367 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850220693716729856 |
|---|---|
| author | Samuchaya Niemrungruang Orathai Sawatdichaikul Siriporn Tanjor Dalad Siriwan |
| author_facet | Samuchaya Niemrungruang Orathai Sawatdichaikul Siriporn Tanjor Dalad Siriwan |
| author_sort | Samuchaya Niemrungruang |
| collection | DOAJ |
| description | Okra is the well-known vegetable, green pods, within the Asian cuisine. This study aimed to evaluate the antioxidant activities of okra seed protein hydrolysate produced by combining three enzymes (trypsin, chymotrypsin, and pepsin). The hydrolysate was filtered through an ultrafiltration membrane with a selective molecular weight of 3 kDa and the % yield was calculated. Subsequently, it was desalted by solid phase extraction (SPE), and peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The results indicated that the protein extracted from defatted okra seed showed a % yield of about 24.25 ± 0.25 %. The % yield of ultrafiltered peptides (≤ 3 kDa) was 14.39 ± 0.85 %. These peptides (after SPE desalting) showed DPPH radical scavenging properties (IC50 0.37 ± 0.79 mg/mL). Moreover, after HILIC fractionation, the HILIC 70 % ACN+0.1 %FA fraction exhibited the most potent DPPH radical scavenging activity (IC50 0.56 mg/mL). This fraction underwent additional separation with RP-HPLC, revealing that fraction 3 (RP-HPLC_3) had the highest DPPH radical scavenging activity, and LC-MS/MS identified its pure peptides. 11 peptides sequence contained hydrophobic, hydrophilic, basic amino acids and aromatic acids residues, which play a crucial role in the antioxidant activity of peptides. According to the cell model, non-cytotoxic concentrations (0.0001 – 1 mg/mL) of peptides from okra seed protein hydrolysates effectively inhibited intracellular ROS generation by LPS-induced oxidative stress in macrophage RAW 264.7 cells, as monitored by DCFH-DA fluorescence. Furthermore, the peptides from okra seed protein hydrolysates at 0.0001 – 1 mg/mL concentrations also strongly suppressed nitric oxide (NO) production. Therefore, the study suggests that peptides from okra seed protein hydrolysates are a potential source of natural antioxidants with pharmaceutical properties worthwhile developing into natural chemo-preventive or nutraceutical products in the future. |
| format | Article |
| id | doaj-art-d7baf5e08857413e8af4af6208b755bb |
| institution | OA Journals |
| issn | 2772-5022 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Applied Food Research |
| spelling | doaj-art-d7baf5e08857413e8af4af6208b755bb2025-08-20T02:06:58ZengElsevierApplied Food Research2772-50222025-06-015110092810.1016/j.afres.2025.100928Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cellsSamuchaya Niemrungruang0Orathai Sawatdichaikul1Siriporn Tanjor2Dalad Siriwan3Department of Food Chemistry and Physics, Institute of Food Research and Product Development, Kasetsart University, 50 Ngamwongwan Road, Ladyao, Chatuchak, Bangkok 10900, Thailand; Corresponding author.Department of Nutrition and Health, Institute of Food Research and Product Development, Kasetsart University, 50 Ngamwongwan Road, Ladyao, Chatuchak, Bangkok 10900, ThailandDepartment of Nutrition and Health, Institute of Food Research and Product Development, Kasetsart University, 50 Ngamwongwan Road, Ladyao, Chatuchak, Bangkok 10900, ThailandDepartment of Nutrition and Health, Institute of Food Research and Product Development, Kasetsart University, 50 Ngamwongwan Road, Ladyao, Chatuchak, Bangkok 10900, ThailandOkra is the well-known vegetable, green pods, within the Asian cuisine. This study aimed to evaluate the antioxidant activities of okra seed protein hydrolysate produced by combining three enzymes (trypsin, chymotrypsin, and pepsin). The hydrolysate was filtered through an ultrafiltration membrane with a selective molecular weight of 3 kDa and the % yield was calculated. Subsequently, it was desalted by solid phase extraction (SPE), and peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The results indicated that the protein extracted from defatted okra seed showed a % yield of about 24.25 ± 0.25 %. The % yield of ultrafiltered peptides (≤ 3 kDa) was 14.39 ± 0.85 %. These peptides (after SPE desalting) showed DPPH radical scavenging properties (IC50 0.37 ± 0.79 mg/mL). Moreover, after HILIC fractionation, the HILIC 70 % ACN+0.1 %FA fraction exhibited the most potent DPPH radical scavenging activity (IC50 0.56 mg/mL). This fraction underwent additional separation with RP-HPLC, revealing that fraction 3 (RP-HPLC_3) had the highest DPPH radical scavenging activity, and LC-MS/MS identified its pure peptides. 11 peptides sequence contained hydrophobic, hydrophilic, basic amino acids and aromatic acids residues, which play a crucial role in the antioxidant activity of peptides. According to the cell model, non-cytotoxic concentrations (0.0001 – 1 mg/mL) of peptides from okra seed protein hydrolysates effectively inhibited intracellular ROS generation by LPS-induced oxidative stress in macrophage RAW 264.7 cells, as monitored by DCFH-DA fluorescence. Furthermore, the peptides from okra seed protein hydrolysates at 0.0001 – 1 mg/mL concentrations also strongly suppressed nitric oxide (NO) production. Therefore, the study suggests that peptides from okra seed protein hydrolysates are a potential source of natural antioxidants with pharmaceutical properties worthwhile developing into natural chemo-preventive or nutraceutical products in the future.http://www.sciencedirect.com/science/article/pii/S2772502225002367Peptides from okra seed protein hydrolysatesAntioxidantPurifiedNitric oxide productionRAW 264.7 cells |
| spellingShingle | Samuchaya Niemrungruang Orathai Sawatdichaikul Siriporn Tanjor Dalad Siriwan Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells Applied Food Research Peptides from okra seed protein hydrolysates Antioxidant Purified Nitric oxide production RAW 264.7 cells |
| title | Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells |
| title_full | Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells |
| title_fullStr | Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells |
| title_full_unstemmed | Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells |
| title_short | Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells |
| title_sort | bioassay guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in lps stimulated raw 264 7 cells |
| topic | Peptides from okra seed protein hydrolysates Antioxidant Purified Nitric oxide production RAW 264.7 cells |
| url | http://www.sciencedirect.com/science/article/pii/S2772502225002367 |
| work_keys_str_mv | AT samuchayaniemrungruang bioassayguidedfractionationofokraseedpeptideonantioxidantactivitiesandinhibitionofoxidativestressinlpsstimulatedraw2647cells AT orathaisawatdichaikul bioassayguidedfractionationofokraseedpeptideonantioxidantactivitiesandinhibitionofoxidativestressinlpsstimulatedraw2647cells AT siriporntanjor bioassayguidedfractionationofokraseedpeptideonantioxidantactivitiesandinhibitionofoxidativestressinlpsstimulatedraw2647cells AT daladsiriwan bioassayguidedfractionationofokraseedpeptideonantioxidantactivitiesandinhibitionofoxidativestressinlpsstimulatedraw2647cells |