本文關(guān)鍵詞： ACE 抑制肽 ACE 抑制蛋白 抑制類型 原發(fā)性高血壓大鼠 松茸 三色雷蘑　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：高血壓是影響人類健康的“無形殺手”,對高血壓疾病的防治及治療藥物的研究是全球醫(yī)學(xué)界面臨的一項(xiàng)艱巨任務(wù)。血管緊張素轉(zhuǎn)換酶(ACE)是腎素-血管緊張素(RAS)和激肽釋放酶-激肽(KKS)這兩個(gè)血壓調(diào)控系統(tǒng)中起關(guān)鍵作用的酶。因此尋找安全性高且副作用小的食源性ACE抑制劑(ACEI)藥物成為一個(gè)熱門課題。食用菌不僅味道鮮美可口、營養(yǎng)豐富,還具有很高的藥用價(jià)值,因此對食用菌中ACEI藥物的研究有著廣泛的應(yīng)用前景。本研究分別從松茸和三色雷蘑的子實(shí)體中分離純化得到一種ACE抑制肽TMP和一種ACE抑制蛋白LTP,并且對其結(jié)構(gòu)、生理生化特性及生物學(xué)活性進(jìn)行了研究。綜合運(yùn)用加熱提取、超濾、離子交換層析及凝膠過濾層析的分離純化手段,從松茸子實(shí)體中得到一個(gè)具有較高ACE抑制活性的肽段,命名為TMP,并且通過LC-MS/MS質(zhì)譜分析得到其氨基酸序列為WALKGYK,分子量為865.04 Da,對ACE的IC50值為0.4μM。TMP在40-90℃具有良好的溫度穩(wěn)定性,90℃條件下孵育2 h對ACE仍有80%的抑制活性。在pH 2.0-11.0范圍內(nèi)也具有較好的穩(wěn)定性,在pH 2.0的酸性條件下,仍然保留40%的ACE抑制活性。通過Lineweaver-Burk動力學(xué)模型測試,TMP屬于非競爭性抑制劑。TMP在10mg/kg時(shí)表現(xiàn)出50%的DPPH自由基清除活性,20 mg/mL時(shí)對亞鐵離子有24%的螯合能力。在單劑量短期原發(fā)性高血壓大鼠(SHR)的體內(nèi)降血壓實(shí)驗(yàn)中,TMP和松茸水提物對SHR大鼠都具有降血壓效果,其有效作用劑量分別是25 mg/kg和400 mg/kg,對SHR大鼠收縮壓的降幅分別是18 mmHg和36 mmHgoTMP劑量為50 mg/kg時(shí),對SHR大鼠收縮壓的降幅為36 mmHg. TMP和松茸子實(shí)體水提物對大鼠的心率都沒有影響。因此松茸及松茸ACE抑制肽TMP的研究為松茸開發(fā)成降血壓功能性食品提供了理論依據(jù)。采用10 kDa超濾膜超濾、兩次強(qiáng)陰離子交換柱層析及凝膠過濾層析的純化技術(shù),從三色雷蘑子實(shí)體中分離純化出一個(gè)分子量為86 kDa的雙亞基ACE抑制蛋白,命名為LTP,純化倍數(shù)為12.7倍,回收率為0.9%,LTP對ACE的IC50值為1.64 mg/mL, N端序列為DGPTMHRQAVADFKQ,疏水性氨基酸比例為40%。采用LC-MS/MS質(zhì)譜技術(shù)得到7條可信度較高的肽段氨基酸序列,并且這7條肽段的疏水性氨基酸殘基所占比例最少為60%。LTP在pH 9.0的堿性環(huán)境孵育2 h后對ACE的抑制活性只有30%,在pH 2.0的酸性環(huán)境下孵育2 h后仍保留有60%的ACE抑制活性。LTP在不同溫度下孵育2 h后,對ACE的抑制活性隨溫度的升高而逐漸降低,溫度穩(wěn)定性差。通過動力學(xué)參數(shù)Km和Vmax的變化及Lineweaver-Burk曲線測試,LTP屬于競爭性抑制劑。三色雷蘑水提物和三色雷蘑ACE抑制蛋白LTP在200 mg/kg及100 mg/kg的劑量下,對SHR大鼠的收縮壓都有顯著的降低作用,其降幅分別是34 mmHg和43 mmHg,并且對大鼠的心率都沒有影響。因此對三色雷蘑ACE抑制劑的研究為此食用菌的進(jìn)一步開發(fā)利用提供了實(shí)驗(yàn)數(shù)據(jù)。
[Abstract]:Hypertension is an "invisible killer" affecting human health. The research on the prevention and treatment of hypertension is a difficult task facing the global medical community. ACE-ACE-RAS-renin-angiotensin (Ras) and angiotensin (ACE). Kallikrein (KKS) is a key enzyme in two blood pressure regulation systems. Therefore, it has become a hot topic to search for food source ACE inhibitor (ACEI) with high safety and small side effects. Edible fungi are not only delicious, but also delicious. It is rich in nutrition and has high medicinal value. In this study, a ACE inhibitory peptide TMP and a ACE inhibitor protein TMP were isolated from the fruiting bodies of Tricholoma tricolor and Tricholoma matsutake, respectively. The physiological and biochemical characteristics and biological activities of Tricholoma matsutake were studied. By means of heating extraction, ultrafiltration, ion exchange chromatography and gel filtration chromatography, a peptide with high ACE inhibitory activity was obtained from the fruit body of Tricholoma matsutake. Its amino acid sequence was WALKGYK with molecular weight 865.04 Da. the IC50 value of ACE was 0.4 渭 M. TMP with good temperature stability at 40-90 鈩，

本文編號：1500870