Sutopo, Christoper Caesar Yudho (2019) Screening Of Bioactive Peptide With Angiotensin-I Converting Enzyme Inhibitory Activity Derived From Enzymatic Hydrolysate Of Black Cumin (Nigella Sativa) Seeds Protein. Magister thesis, Universitas Brawijaya.

Indonesian Abstract


English Abstract

Bioactive peptide with ACE inhibitory activity has been interested many researchers due to their potential as antihypertensive, safer and milder alternatives than synthetic drugs. Nigella sativa seeds are used for edible and medicinal purposes in many countries, including Egypt, Syria, Iran and to a slight extent in Tunisia. The seeds oil has been reported to possess antitumor activity, antioxidant activity, anti-inflammatory activity, antibacterial activity and a stimulatory effect on the immune system. The seeds have also been used as diuretics, anti-hypertensive, muscle relaxants and as immunity enhancers in immune-compromised people. Importantly, the seeds have been reported to be safe when used orally with a moderate amount in food. The objective of this study is to isolate a novel bioactive peptide with ACE inhibitory activity derived from Nigella sativa seeds protein hydrolysate. Firstly, black cumin seed protein was hydrolyzed with pepsin, trypsin and α-chymotrypsin. Over the ultrafiltration using <3 kDa Molecular weight Cut Off and Solid Phase IV Extraction C-18 column the hydrolysate from α-chymotrypsin showed the most potential ACE inhibitory activity with IC50 value 34.4 ± 1.5 μg/mL and the highest degree of hydrolysis was 10.8%. Two orthogonal bioassay-guided fractionations (RP-HPLC and Offline-SCX) were simultaneously applied to screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from black cumin seeds protein (BCSP) hydrolysate. The peptides in the most potential RP-HPLC and Offline-SCX fraction were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) respectively. VTPVGVPKW (VW-9) was identified as the most effective ACEI peptide, found in the most active RP-HPLC and Offline-SCX fractions. It was found that VW-9 indicate a pro-drugs type caused by the inhibitory activity of the peptide was increased from IC50 1.8 ± 0.09 μM (without ACE pre-incubation) into 1.3 ± 0.06 μM after incubation with ACE. Moreover, the kinetic study revealed that VW-9 is a non-competitive inhibitor and its molecular docking interaction toward ACE was proposed. Therefore, we can conclude this is the first report about bioactive peptide derived from Nigella sativa seeds protein. It may be beneficial for preventing hypertension and functional food development.

Other Language Abstract

具有ACE抑制效果的生物活性肽作為替代藥物,比起合成藥物讓研究人員關注是因其潛在的抗高血壓更安全且溫和。許多國家用食用和藥用Nigella sativa種子(黑種子),包含埃及、敘利亞、伊朗和一部分的突尼西亞。據報導,種子油具有抗腫瘤活性、抗氧化活性、抗發炎活性、抗菌活性和對免疫系統的刺激作用。種子也被用作利尿劑,抗高血壓,肌肉鬆弛劑和免疫受損人群的免疫增強劑。重要的是,據報導,當在食物中添加適量種子食用是安全的。本研究的目的是分離源自黑種子蛋白水解物具有ACE抑制活性的新型生物活性肽。首先,用胃蛋白酶、胰蛋白酶和α-胰凝乳蛋白酶水解黑種子蛋白。在使用<3kDa分子量截留和固相萃取C18管柱的超濾中,來自α-胰凝乳蛋白酶的水解產物顯示出最具潛力的ACE抑制活性,IC50值為34.4±1.5μM,最高水解度為10.8%。逆向高效能液相層析儀(RP-HPLC)和強陽離子交換層析(SCX)兩種正交分群同時用於篩選來自黑種子蛋白(BCSP)水解產物抑制血管收縮素轉化酶I(ACEI)的胜肽。具有最高活性的HPLC和SCX分液分別通過液相層析-串聯式質譜儀(LC-MS/MS),VTPVGVPKW(VW具有ACE抑制效果的生物活性肽作為替代藥物,比起合成藥物讓研究人員關注是因其潛在的抗高血壓更安全且溫和。許多國家用食用和藥用Nigella sativa種子(黑種子),包含埃及、敘利亞、伊朗和一部分的突尼西亞。據報導,種子油具有抗腫瘤活性、抗氧化活性、抗發炎活性、抗菌活性和對免疫系統的刺激作用。種子也被用作利尿劑,抗高血壓,肌肉鬆弛劑和免疫受損人群的免疫增強劑。重要的是,據報導,當在食物中添加適量種子食用是安全的。本研究的目的是分離源自黑種子蛋白水解物具有ACE抑制活性的新型生物活性肽。首先,用胃蛋白酶、胰蛋白酶和α-胰凝乳蛋白酶水解黑種子蛋白。在使用<3kDa分子量截留和固相萃取C18管柱的超濾中,來自α-胰凝乳蛋白酶的水解產物顯示出最具潛力的ACE抑制活性,IC50值為34.4±1.5μM,最高水解度為10.8%。逆向高效能液相層析儀(RP-HPLC)和強陽離子交換層析(SCX)兩種正交分群同時用於篩選來自黑種子蛋白(BCSP)水解產物抑制血管收縮素轉化酶I(ACEI)的胜肽。具有最高活性的HPLC和SCX分液分別通過液相層析-串聯式質譜儀(LC-MS/MS),VTPVGVPKW(VW-9)被鑑定為最具有ACEI肽,存在於具有最高活性的HPLC和SCX分液中。VW-9在與ACE預混和後,胜肽的抑制活性IC50 1.8±0.09降低至1.3±0.06μM可得知為前驅藥類型。此外,酵素動力學研究表明 II VW-9是一種非競爭性抑製劑,並提出了它與ACE的分子架接相互作用。因此,我們可以得出結論,這是源自Nigella sativa黑種子蛋白的第一份生物活性肽報告,它可能有益於預防高血壓和功能性食品的發展。-9)被鑑定為最具有ACEI肽,存在於具有最高活性的HPLC和SCX分液中。VW-9在與ACE預混和後,胜肽的抑制活性IC50 1.8±0.09降低至1.3±0.06μM可得知為前驅藥類型。此外,酵素動力學研究表明 II VW-9是一種非競爭性抑製劑,並提出了它與ACE的分子架接相互作用。因此,我們可以得出結論,這是源自Nigella sativa黑種子蛋白的第一份生物活性肽報告,它可能有益於預防高血壓和功能性食品的發展。

Item Type: Thesis (Magister)
Identification Number: TES/572.65/SUT/s/2019/041902955
Subjects: 500 Natural sciences and mathematics > 572 Biochemistry > 572.6 Proteins > 572.65 Components of proteins
Divisions: S2 / S3 > Magister Keteknikan Pertanian, Fakultas Teknologi Pertanian
Depositing User: Endang Susworini
URI: http://repository.ub.ac.id/id/eprint/177604
Christoper Caesar (2).pdf
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