TW201043239A - An isolated peptide and the preparation processes and applications thereof - Google Patents

An isolated peptide and the preparation processes and applications thereof Download PDF

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TW201043239A
TW201043239A TW98119377A TW98119377A TW201043239A TW 201043239 A TW201043239 A TW 201043239A TW 98119377 A TW98119377 A TW 98119377A TW 98119377 A TW98119377 A TW 98119377A TW 201043239 A TW201043239 A TW 201043239A
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peptide
treatment
chlorella
algae
activity
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TWI369211B (en
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Tony Ji Fang
I-Chuan Sheih
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Nat Univ Chung Hsing
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Abstract

Disclosed herein is a peptide isolated from an algae of Chlorella genus, which has angiotensin I-converting enzyme inhibitory activity, anti-oxidative activity, anti-inflammation activity, and activity of inhibiting cancer cell proliferation. Also disclosed are the preparation processes and application of said peptide.

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201043239 六、發明說明： 【發明所屬之技術領域】 本發明是有關於一種分離自小球藻屬藻類（al§ae 〇f C/i/ore"a genus)的肽（peptide)，它具有血管緊縮素I-轉化酵 素（angiotensin I-converting enzyme)抑制活性、抗氧化活性 Λ 、抗發炎活性以及抑制癌細胞（特別是胃腺癌細胞）增生的活 _ 性。本發明亦有關於該肽的製備方法與應用。 【先前技術】 〇 血管緊縮素I-轉化酵素（angiotensin I-converting enzyme, ACE)在負責調節血壓的腎素-血管緊縮素系統 (renin-angiotensin system, RAS)中扮演一個重要的角色。 ACE可將不活化的血管緊縮素I轉化成具有強力的血管收 縮效用（vasoconstrictive effect)之血管緊縮素II，並且將具 有舒張血管功能的舒緩素（bradykinin)去活化，因而造成血 管收縮以及血壓增高。 • 目前ACE的抑制劑[諸如依那普利（enalapril)以及卡托 普利（captopril)]已經被使用作為抗高血壓的藥物（C. Chevillard ei «/· (1988), «/· _P/mrmaco/·，147:23-28)。然 而，這些合成的ACE抑制劑在臨床上會造成許多非所欲的 副作用，諸如咳漱（cough)、味覺喪失（lost of taste)、腎損傷 (renal impairment)以及血管神經性水腫（angioneurotic edema)(T.F. Antonios and G.A. MacGregor (1995), J.201043239 VI. Description of the Invention: [Technical Field] The present invention relates to a peptide isolated from a genus Chlorella (al§ae 〇f C/i/ore" a genus) having a blood vessel Angiotensin I-converting enzyme inhibits activity, antioxidant activity, anti-inflammatory activity, and inhibits the proliferation of cancer cells, particularly gastric adenocarcinoma cells. The invention also relates to a preparation method and application of the peptide. [Prior Art] ang Angiotensin I-converting enzyme (ACE) plays an important role in the renin-angiotensin system (RAS), which regulates blood pressure. ACE converts inactivated angiotensin I into angiotensin II with potent vasoconstrictive effect and deactivates bradykinin with diastolic vascular function, resulting in vasoconstriction and increased blood pressure . • Current ACE inhibitors [such as enalapril and captopril] have been used as antihypertensive drugs (C. Chevillard ei «/· (1988), «/· _P/ Mrmaco/·, 147: 23-28). However, these synthetic ACE inhibitors cause many undesired side effects in the clinic, such as cough, lost of taste, renal impairment, and angioneurotic edema. (TF Antonios and GA MacGregor (1995), J.

Sm/?/?/·，13:S11-S16)。因此，當今醫藥界已嘗試 從天然的來源中找尋具有ACE抑制活性的成分來供醫療所 3 201043239 需。 在好氧生物體（aerobic organisms)進行呼吸反應 (respiration)的過程中會形成促氧化劑（prooxidants)，亦即活 性氧族（reactive oxygen species, ROS)以及自由基（free redicals)，它們非常的不穩定並且會快速地與體内的基團或 物質反應，進而導致細胞或組織的氧化性傷害（oxidative damage)。當促氧化劑的形成與抗氧化劑（antioxidant)的能力 之間的恆定被破壞時，氧化性傷害會被累積並且造成病理生 理學現象（pathophysiological events)。有文獻報導指出，活 性氧族以及自由基會攻擊重要的生物分子（諸如DNA、蛋白 質以及脂質）而導致退化性疾病狀況（degenerative disease conditions)，例如：癌症、胃潰瘍（gastric ulcers)、阿茲海默 症（Alzheimer’s Disease)、關節炎（arthritis)以及缺血性再灌 流（ischemic reperfusion)(K.P. Suja W α/· (2004)，·/. JgWc. Food Chem., 52:912-915 ； P. Leanderson et al. (1997), Free Radic. Biol. Med., 23:235-242 ； D. Debashis et al. (1997), Free 及aAc· 5b/· Mec?·, 23:8-18 ; W&O.Vajraguptaeia/· (2000)，Mec?· CAem·，8:2617-2628)。為 了維持促氧化 劑-抗氧化劑平衡（prooxidant-antioxidant balance)，自由基以 及ROS的移除可能是一活體在對抗各種疾病時最有效的防 禦之一。 目前許多合成的抗氧化劑[諸如丁基羥基甲氧苯 (butylated hydroxyanisole，BHA)、二丁基經基甲苯（dibutyl hydroxytoluene, BHT)以及沒食子酸丙 S旨（propyl gallate, PG)] 201043239 已經被用來延遲氧化過程（oxidation process)。然而，合成 的抗氧化劑之使用必須受到嚴格的控管以避免潛在的健康 危害（P.J. Park ei ct/· (2001)，乂 c/jem. Soc·，78:651- 656)。因此’本技藝仍然存在有一需要來發展出可以有效地 延遲氧化過程並且不會產生非所欲的副作用的藥物。 • 藻類（algae)因為在地球上的含量豐富而已成為人類重要 ^ 的食物來源之一。在各種已知的食用藻類當中，小球藻屬 的藻類（algae of C/i/ore//a genus)含有豐富的蛋白質以及人體 〇 無法自行合成的胺基酸，因此特別受到注意並且廣泛地被 使用作為天然保健食品的原料。小球藻屬的藻類是一種呈 球狀且直徑約為2至10 μιη的單細胞綠藻（single-cell green algae)，它是屬於小球藻目（Chlorellales)、小球藻科 (Chlorellaceae)、小球藻屬（CA/ore//a)，主要生長在淡水 (freshwater)。常見的小球藻屬藻類包括：小球藻（CA/ore/k vw/gan\s)、蛋白核小球藻（CA/ore//a 、橢圓小球 藻(Chlorella ellipsoidea) ' 微小小球藻(Chlorella 〇 /ni'wwi&W/wa)以及对熱性小球藻（CA/ore//a sorohWawa)。 近年來，經由各種食物蛋白質（food proteins)的酵素水 解（enzymatic hydrolysis)而被分離出的肽（peptides)已被證實 具有許多生物活性，例如：免疫調節（immunomodulation)、 抗氧化（antioxidation)、抗高血壓（antihypertension)、抗癌 (anticancer)，以及抗微生物活性（antimicrobial activity)等。 已有文獻報導可從藻類的酵素水解產物（enzymatic hydrolysates)中分離出具有生物活性的肽。在K. Suetsuna 5 201043239 and T. Nakano (2000)，·/· 沿oc々em·，11:450-454 中，Κ.Sm/?/?/·, 13:S11-S16). Therefore, the medical community has tried to find ingredients with ACE inhibitory activity from natural sources for medical institutions. In the process of respiration of aerobic organisms, prooxidants, namely reactive oxygen species (ROS) and free redicals, are formed. Stable and rapidly reacts with groups or substances in the body, which in turn leads to oxidative damage to cells or tissues. When the constant between the formation of an oxidizing agent and the ability of an antioxidant is destroyed, oxidative damage is accumulated and causes pathophysiological events. It has been reported in the literature that reactive oxygen species and free radicals attack important biomolecules (such as DNA, proteins and lipids) leading to degenerative disease conditions such as cancer, gastric ulcers, Azhai. Alzheimer's Disease, arthritis, and ischemic reperfusion (KP Suja W α/· (2004),··. JgWc. Food Chem., 52:912-915; P. Leanderson et al. (1997), Free Radic. Biol. Med., 23:235-242; D. Debashis et al. (1997), Free and aAc·5b/· Mec?, 23:8-18; W&amp O.Vajraguptaeia/· (2000), Mec?· CAem·, 8:2617-2628). In order to maintain a pro-antantant-antioxidant balance, free radicals and ROS removal may be one of the most effective defenses against a variety of diseases in vivo. Many synthetic antioxidants [such as butylated hydroxyanisole (BHA), dibutyl hydroxytoluene (BHT), and propyl gallate (PG)] 201043239 have been Used to delay the oxidation process. However, the use of synthetic antioxidants must be strictly controlled to avoid potential health hazards (P.J. Park ei ct/. (2001), 乂 c/jem. Soc., 78:651-656). Therefore, there is still a need in the art to develop a drug that can effectively delay the oxidation process without causing undesirable side effects. • Algae has become one of the most important sources of food for humans because of its rich content on Earth. Among various known edible algae, the algae of algae (algae of C/i/ore//a genus) is rich in protein and amino acids which are not synthesized by the human body, so it is particularly noticed and widely used. It is used as a raw material for natural health foods. The algae of the genus Chlorella is a spheroidal single-cell green algae having a diameter of about 2 to 10 μηη, belonging to the family Chlorellales and Chlorellaceae. Chlorella (CA/ore//a), mainly grown in freshwater. Common chlorella algae include: Chlorella (CA/ore/k vw/gan\s), Chlorella pyrenoids (CA/ore//a, Chlorella ellipsoidea) tiny balls Algae (Chlorella 〇/ni'wwi & W/wa) and Thermococci (CA/ore//a sorohWawa). In recent years, they have been separated by enzymatic hydrolysis of various food proteins. The peptides have been shown to have many biological activities, such as: immunomodulation, antioxidation, antihypertension, anticancer, and antimicrobial activity. It has been reported in the literature that biologically active peptides can be isolated from enzymatic hydrolysates of algae. In K. Suetsuna 5 201043239 and T. Nakano (2000),··· along oc々em·, 11: 450-454, Κ.

Suetsuna與T. Nakano藉由離子交換層析法（ion exchange chromatography)與凝膠過滤法（gel filtration)而從裙帶菜 (wakame)((/«i/flWa 的胃酶消化物（peptic digest) 中分離出具有 ACE抑制活性的肽分離部分（peptide fractions)。該等肽分離部分進一步在一逆相管柱（reverse phase column)上被層析，藉此而得到4個具有ACE抑制活 性的四肽（tetrapeptides)。實驗結果顯示：自發性高血壓大 鼠（spontaneously hypertensive rats, SHRs)在被 口服投藥以該 等四肽之後，血壓被顯著地降低。 K. Suetsuna and J.R. Chen (2001), Mar. Biotechnol., 3:305-309中，K. Suetsuna與J.R. Chen藉由離子交換層析 法與凝膠過滤法分別從小球藻（C7?/ore//a vw/gaW·?)以及鈍頂 螺旋藻的胃酶消化物中分離出會抑制 ACE的肽分離部分（peptidic fractions)。經由實驗發現，將 該等肽分離部分（劑量為200 mg/kg體重）口服投藥至自發性 高血壓大鼠中會造成顯著的抗高血壓效用。因此，K. Suetsuna與J.R. Chen進一步從該等肽分離部分中分離出10 個活性肽（active peptides)，其中有5個是得自於小球藻，而 有5個是得自於鈍頂螺旋藻。 M. Sato et al. (2002), J. Agric. Food Chem., 50:6245-6252中，M. Sato等人藉由使用在一逆相管柱上的三階段高 效能液相層析法（three-step high-performance liquid chromatography)而從裙帶菜的蛋白酶S “Amano”消化物 201043239 (Protease S Amano” digest of wakame)中分離出 7 個具有 ACE抑制活性的肽。實驗結果顯示，分離出的肽具有在活 體外對抗胃腸道蛋白酶的抗性。此外，自發性高血壓大鼠 被單一口服投藥以各個肽之後，該等肽被確定具有抗高血 壓效用。 另一方面，在藻類精華（algae essence)的製造過程中會 產生大ΐ的藻類廢棄物（algae wastes)，而在這些藻類廢棄物 中仍含有大量的蛋白質，若是可以利用這些藻類廢棄物來 開發出具有生物活性的肽，不但可以降低生產成本，同時 也可讓資源被有效地再利用。但是就申請人所知，迄今尚 …、任何文獻資料或專利前案曾經揭示藻類廢棄物可被用來 開發具有生物活性的肽。 雖然已有上述的研究，從藻類和/或其廢棄物中開發出 具有產業價值之新產物是相關研究人員所致力的目標。經 研究，申請人意外地從小球藻（C/i/ore//a …）的廢棄物 中分離出一具有生物活性的肽。因此，該具有生物活性的 肽被預期可供作為調節生理機能的保健食品或醫藥品。 【發明内容】 發明概要 於是，在第一個方面，本發明提供一種具有一如序列 辨識編號：1所示的胺基酸序列之經分離的肽，它是藉由一 包含下列步驟之方法而被製得： 提供一種屬於一小球藻屬藻類（31§狀〇f genus) 的藻類材料； 7 201043239 將該藻類材料進行—使用一蛋白酶的水解處理，藉此 而得到一水解物；以及 稽此* 將該水解物進行-純化處理，藉此而得到該狀。 依據本發明之分離自小球藻屬藻類UlgaeGf — 奸刪）的肽被證實W管緊縮素ι-轉化酵素（anglGtensin , converting enZyme)抑制活性、抗氧化活性、抗發炎活性以 及抑制癌細胞（特別是胃腺癌細胞）增生的活性。 因此，在第二個方面，本發明提供—種藥學組成物或 食品產品伽d Pn)duet)’其包含有—如上所述的肽以作為 -活性：份。依據本發明的藥學組成物或食品產品被預期 具有抗高血壓、抗氧化、抗發炎以及抗癌之功效。 本發明的上述以及其它目的、特徵與優點，在參照以 下的詳細說明與較佳實施例和隨文檢附的圖式後， 明顯。 發明的詳細說明 為了這本A明書之目的’將被清楚地瞭解的是：文字“ 包含有（C〇mprising)”意指“包含但不限於，，以及文字“包括 (comprises)”具有—對應的意義。 要被瞭解的是：若有任何一件前案刊物在此被引述， 該前案刊物不構成個下述承認：在台灣或任何其他國家 之中，該前案刊物形成本技藝中㈣見—般知識之一部分 除非另外有所定義，在本文中所使用的所有技術性與 科學術β。具有熟悉本發明所屬技藝的人士所共同瞭解的意 201043239 如本文中所用的，“肽（peptide)”、“寡肽（〇lig0peptide)，， 、“多肽（polypeptide)”和“蛋白質（protein)，’等術語可被相互 交換使用’且意指一種由胺基酸殘基所構成的聚合物，其 中一或多個胺基酸殘基是天然存在的胺基酸（naturally occurring amino acids)或人造化學仿效物（aritificial chemical 在本文中，胺基酸可以其全名、三個字母的縮寫以及 〇 單一子母符號來表示，這是本技藝中已詳知的。此外，依 照肽標示的慣例，蛋白質的左端是N端（胺基端），而右端是 C端（羧基端）。 目前已知的先前研究大多數是從天然的藻類中去分離 出具有各種不同生物活性的肽。 然而，在藻類製品的加工Suetsuna and T. Nakano from wakame ((/«i/flWa peptic digest) by ion exchange chromatography and gel filtration Peptide fractions having ACE inhibitory activity are isolated. The peptide fractions are further chromatographed on a reverse phase column, thereby obtaining four tetrapeptides having ACE inhibitory activity. (tetrapeptides).Experimental results show that spontaneously hypertensive rats (SHRs) have significantly reduced blood pressure after oral administration of these tetrapeptides. K. Suetsuna and JR Chen (2001), Mar. Biotechnol., 3:305-309, K. Suetsuna and JR Chen from chlorella (C7?/ore//a vw/gaW·?) and blunt-top spiral by ion exchange chromatography and gel filtration, respectively Peptidic fractions which inhibit ACE were isolated from the stomach enzyme digest of the algae. It was found through experiments that the peptide fraction (dose of 200 mg/kg body weight) was orally administered to spontaneously hypertensive rats. Will cause significant Antihypertensive effect. Therefore, K. Suetsuna and JR Chen further isolated 10 active peptides from the peptide fractions, of which 5 were derived from chlorella and 5 were obtained. From Spirulina platensis. M. Sato et al. (2002), J. Agric. Food Chem., 50:6245-6252, M. Sato et al. by using three stages on a reverse phase column Seven-step high-performance liquid chromatography and isolation of seven peptides with ACE inhibitory activity from Protease S "Amano" digest 201043239 (Protease S Amano" digest of wakame The experimental results show that the isolated peptide has resistance against gastrointestinal proteases in vitro. In addition, after spontaneously hypertensive rats are administered orally with individual peptides, the peptides are determined to have antihypertensive effects. On the one hand, in the manufacturing process of algae essence, large algae wastes are produced, and in these algae wastes, a large amount of protein is still contained, and if these algae wastes can be used, A biologically active peptide, can not only reduce the production cost, but also allows resources are effectively reused. However, to the best of the applicant's knowledge, any literature or patent preamble has previously revealed that algal waste can be used to develop biologically active peptides. Despite the above studies, the development of new products of industrial value from algae and/or their waste is a goal of the researchers concerned. Upon investigation, the applicant unexpectedly isolated a biologically active peptide from the waste of chlorella (C/i/ore//a ...). Therefore, the biologically active peptide is expected to be useful as a health food or pharmaceutical for regulating physiological functions. SUMMARY OF THE INVENTION Accordingly, in a first aspect, the present invention provides an isolated peptide having an amino acid sequence as shown in Sequence Identification Number: 1, which is obtained by a method comprising the following steps Produced: an algae material belonging to a chlorella algae (31 § 〇f genus); 7 201043239 The algae material is subjected to a hydrolysis treatment using a protease, thereby obtaining a hydrolyzate; This * The hydrolyzate was subjected to a purification treatment, whereby the form was obtained. The peptide isolated from the genus UlgaeGf of the genus Chlorella according to the present invention was confirmed to have inhibitory activity, antioxidative activity, anti-inflammatory activity, and inhibition of cancer cells by the angiotensin-imprinting enzyme (anglGtensin, converting enZyme). It is the activity of proliferation of gastric adenocarcinoma cells. Accordingly, in a second aspect, the present invention provides a pharmaceutical composition or food product gamma Pn)duet) which comprises - a peptide as described above as - active: part. The pharmaceutical composition or food product according to the present invention is expected to have antihypertensive, anti-oxidant, anti-inflammatory, and anti-cancer effects. The above and other objects, features, and advantages of the present invention will be apparent from the description and appended claims appended claims DETAILED DESCRIPTION OF THE INVENTION For the purposes of this A specification, it will be clearly understood that the text "includes (C〇mprising)" means "including but not limited to, and the words "comprises" have - The corresponding meaning. It is to be understood that if any of the previous publications is quoted here, the previous publication does not constitute the following recognition: in Taiwan or any other country, the former publication forms the skill (4) See the general knowledge, unless otherwise defined, all the technical and scientific techniques used herein. As understood by those skilled in the art to which the present invention pertains, 201043239, as used herein, "peptide "peptide", "oligopeptide", "polypeptide" and "protein", and the like may be used interchangeably and mean one consisting of amino acid residues. a polymer wherein one or more amino acid residues are naturally occurring amino acids or artificial chemical analogs (aritificial chemical) This is known in the art by its full name, a three-letter abbreviation, and a single-parent symbol. Furthermore, according to the convention of peptide labeling, the left end of the protein is the N-terminus (amine-based end), and The right end is the C-terminus (carboxyl end). Most of the previously known prior studies have isolated peptides with various biological activities from natural algae. However, in the processing of algae products

於是，本發明提供一種具有一 的胺基酸序列之經分離的肽 方法而被製得： Γ 一如序列辨識編號：1所示 它是藉由一包含下列步驟之Thus, the present invention provides an isolated peptide method having an amino acid sequence as follows: Γ as shown in Sequence Identification Number: 1, which consists of

的藻類材料； 將該藻類材料進行一使用 小球藻屬藻類（algae of genuy 而得到一水解物；以及 將該水解物進行— 一蛋白晦的水解處理，藉此 —純化處理，藉此而得到該肽。 201043239 依據本發明’在該方法中，該小球藻屬藻類是選自於 下列所構成的群組：小球藻vw/gflrb)、蛋白核小 球藻（C/i/㈣//β外㈣〇ΰ/0>5α)、橢圓小球藻（c/j/崎… ellipsoidea)、微小小珠藻（Chlorella minutissima) ' 耐熱性小 球藻（CA/ore//a β)，以及它們的組合。在本發明的 一個較佳具體例中，該小球藻屬藻類是小球藻（c/2/〇^"a vulgaris)。 依據本發明，在該方法中，該藻類材料可以是天然的 材料或經加工處理的材料（pr〇cessed material)(例如經冷凍 乾燥或噴霧乾燥的材料）。 在本發明的一個較佳具體例中，該藻類材料在被進行 水解之前，有先經過-加工處理。該加工處理包括，但不 限於丄水萃取處理、酵素水解處理、熱處理、高溫高慶處 理、高壓均質處理、酸處理、驗處理以及冷;東解凌處理， 材們的組合。在本發明的-個更佳具體例中，該藻類 ^Γ、球藻廢棄物（赠S °f CL如⑷，它是在 二精華（咖essence)的製造過程中，將小球藻（咖㈣ «发叫進行—水萃取處理後所得到的廢棄物。 構成2本發明，在該方法t，該蛋㈣是選自於下列所 構成的群組··胃蛋白酶( 蛋白梅型風味蛋白崎=白酶(心㈣、木瓜 及它們的組合。較佳地，料^崎⑽^ A)，以 轉。在本發明的一個較佳：疋胃蛋白酶或者驗蛋白 。 ㈣具體例中，該蛋白酶是胃蛋白梅 10 201043239 有關使用蛋白酶的水解處理，這是本技術領域中廣泛 被使用的技術。因此，熟習此項技術者可依自身的專業素 養並視發明所需來選定適當的蛋白酶，並據此來決定適當 的反應溫度、pH值，以及作用時間等參數條件，俾以得到 含有本發明的肽的水解物。 - 依據本發明，在該方法中，該水解物在被進行純化處 理之前可先經過一分離處理，藉此可移除非所欲的大分子 物質以及水不溶性成分。該分離處理可以採用熟習此項技 〇 藝者所詳知且慣用的技術，例如，離心或過濾來進行。 依據本發明，在該方法中，該純化處理可以採用熟習 此項技藝者所詳知且慣用的技術來進行。較佳地，該純化 處理是選自於下列所構成的群組：硫酸胺分級分離處理 (ammonium sulfate fractionation)、凝膠過瀘、層析（gel filtration chromatography)、離子交換層析（ion exchange r chromatography)、逆相層析（reverse phase chromatography) 、疏水性交互作用層析（hydrophobic interaction 〇 chromatography)，以及它們的組合。 依據本發明，在該方法中，該肽可藉由任一種已知的 回收方法而被回收。該等已知的方法包括，但不限於：冷 凍乾燥法、喷霧乾燥處理（spray-drying)、蒸發處理 (evaporation)、加熱乾燥處理（heat-drying)，以及它們的組 合0 此外，本發明的肽亦可採用已知且慣用的化學合成方 法或遺傳工程技術而被製備，例如，固相肽合成法（solid 11 201043239 phase peptide synthesis)(Jang et al. (2008), food chemistry, 107:327-336)等。 依據本發明的肽被證實具有血管緊縮素I-轉化酵素 (angiotensin I-converting enzyme)抑制活性、抗氧化活性、 抗發炎活性以及抑制癌細胞（特別是胃腺癌細胞）增生的活性 〇 因此，本發明提供一種藥學組成物，其包含有一如上 所述的肽以作為一 ACE抑制劑。該藥學組成物被預期可供 用於治療或預防與ACE有關聯的疾病，例如：心血管疾病 (cardiovascular disease)、冠狀動脈心臟病（coronary heart disease)、腦血管疾病（cerebrovascular disease)、周邊血管疾 病（peripheral vascular disease)、心律不整（arrhythmia)、高 jk 壓（hypertension)、心肌梗塞（myocardial infract)等。 本發.明亦提供一種藥學組成物，其包含有一如上所述 的肽以作為一抗氧化劑。該藥學組成物被預期可供用於治 療或預防與氧化性傷害有關聯的疾病，例如：大腦缺血 (cerebral ischemia)、糖尿病（diabetes mellitus)、缺血性再灌 流（ischemic reperfusion)、帕金森氏症（Parkinson’s disease) 、休克（shock)、散播性血管内凝血症（disseminated intravascular coagulation syndrome)、動脈粥樣硬化 (atherosclerosis)、神經退化性疾病（neurodegenerative disease)、關節炎（arthritis)、胃潰瘍（gastric ulcers)、阿茲海 默症（Alzheimer’s Disease)、結腸潰癌（ulcerative colitis)、癌 症（cancer)、唐氏症（Down’s syndrome)、黃斑退化（macular 12 201043239 degeneration)等 ° 本發明亦提供一種藥學組成物，其包含有一如上所述 的肽以作為一抗發炎劑。該藥學組成物被預期可供用於治 療或預防發炎性疾病（inflammatory disease)，例如：中風 (stroke)、多發性硬化症（multiple sclerosis)、腦炎 . (encephalitis)、敗血性休克（septic shock)、成人呼吸性窘迫 症候群（adult respiratory distress syndrome)、心肌炎 (myocarditis)、關節炎（arthritis)、神經炎（neuritis)以及腎小 〇 球性腎炎（glomerulonephritis)等。 此外，本發明提供一種藥學組成物，其包含有一如上 所述的肽以作為一用以抑制癌細胞（特別是胃腺癌細胞）的生 長的活性組份。 依據本發明的肽亦被預期到在製備用以抗高血壓、抗 氧化、抗發炎以及抗癌的保健食品或非處方醫藥品上之應 r 用。 【實施方式】 〇 較佳實施例之詳細說明 本發明將就下面的實施例來做進一步說明，但應瞭解 的是，該等實施例僅是供例示說明用，而不應被解釋為本 發明的實施上的限制。 實施例 實驗材料： 1.小球藻廢棄物（waste of C7i/ore//a vw/gari··?)是購自於斗六 養藻場，它是在藻類精華（algae essence)的製造過程中， 13 201043239 將小球藻（C/i/ore//iz vw/garb)進行一水萃取處理之後所得 到的廢棄物。小球藻廢棄物在進行下面的實驗之前被熱 風乾燥並且被保存於-20°C下備用。 2_ 蛋白酶（protease) : A 型風味蛋白酶（flavourzyme type A) 以及驗蛋白酶（alcalase)是購自 Novo Nordisk A/S (Copenhagen，Denmark);木瓜蛋白酶（papain)是購自 Amano (Nagoya, Japan);胃蛋白酶（pepsin)是購自 Nacalai Tesque (Kyoto, Japan);以及胰酶（pancreatin)是購自 Sigma Chemical Co_ (St. Louis, MO，USA)。Algae material; the algae material is subjected to a hydrolysate using algae of genuy; and the hydrolyzate is subjected to hydrolysis treatment of a peptone, thereby obtaining a purification treatment, thereby obtaining The peptide. 201043239 According to the invention 'in the method, the Chlorella sp. is selected from the group consisting of: Chlorella vw/gflrb), Chlorella pyrenoids (C/i/(4)/ /β outside (four) 〇ΰ / 0> 5α), ellipsoidella (c / j / s... ellipsoidea), Chlorella minutissima ' heat-resistant chlorella (CA / ore / / a β), And their combination. In a preferred embodiment of the invention, the chlorella algae is chlorella (c/2/〇^" a vulgaris). According to the invention, in the method, the algal material may be a natural material or a pr〇cessed material (e.g., a freeze-dried or spray-dried material). In a preferred embodiment of the invention, the algal material is subjected to a processing prior to being hydrolyzed. The processing includes, but is not limited to, hydrophobic water extraction treatment, enzyme hydrolysis treatment, heat treatment, high temperature treatment, high pressure homogenization treatment, acid treatment, inspection treatment, and cold; East Jieling treatment, a combination of materials. In a more specific embodiment of the present invention, the algae and the algae waste (send S °f CL as (4), it is in the manufacturing process of the second essence (caessence), the chlorella (cafe) (4) «Distribution - Waste obtained after water extraction treatment. Composition 2 The present invention, in the method t, the egg (4) is selected from the group consisting of pepsin (protein plum flavor protein protein = white enzyme (heart (four), papaya and combinations thereof. Preferably, the material is saki (10) ^ A), in order to rotate. In a preferred embodiment of the invention: chymotrypsin or protein assay. (d) In a specific example, the protease Is a gastric protein plum 201043239. Regarding the hydrolysis treatment using protease, this is a technique widely used in the art. Therefore, those skilled in the art can select an appropriate protease according to their own professional literacy and according to the needs of the invention, and Accordingly, the reaction conditions such as the appropriate reaction temperature, pH value, and action time are determined to obtain a hydrolyzate containing the peptide of the present invention. - According to the present invention, in the method, the hydrolyzate is subjected to purification treatment before being subjected to purification treatment Can pass a separation The treatment whereby the undesired macromolecular material and the water-insoluble component can be removed. The separation treatment can be carried out by techniques well known and customary to those skilled in the art, such as centrifugation or filtration. In this method, the purification treatment can be carried out by a technique known and customary to those skilled in the art. Preferably, the purification treatment is selected from the group consisting of: ammonium sulfate fractionation treatment ( Ammonium sulfate fractionation), gel filtration chromatography, ion exchange r chromatography, reverse phase chromatography, hydrophobic interaction 〇chromatography And a combination thereof. According to the invention, in the method, the peptide can be recovered by any known recovery method, including, but not limited to, freeze drying, spraying Spray-drying, evaporation, heat-drying, and combinations thereof Furthermore, the peptides of the present invention can also be prepared by known and conventional chemical synthesis methods or genetic engineering techniques, for example, solid phase peptide synthesis (solid 11 201043239 phase peptide synthesis) (Jang et al. (2008), food Chemistry, 107:327-336), etc. The peptide according to the present invention has been confirmed to have angiotensin I-converting enzyme inhibitory activity, antioxidant activity, anti-inflammatory activity, and inhibition of cancer cells (especially gastric glands). Active activity of cancer cells) Accordingly, the present invention provides a pharmaceutical composition comprising a peptide as described above as an ACE inhibitor. The pharmaceutical composition is expected to be useful for the treatment or prevention of diseases associated with ACE, such as: cardiovascular disease, coronary heart disease, cerebrovascular disease, peripheral vascular disease (peripheral vascular disease), arrhythmia, hyperthorax, myocardial infarction, etc. The present invention also provides a pharmaceutical composition comprising a peptide as described above as an antioxidant. The pharmaceutical composition is expected to be useful for treating or preventing diseases associated with oxidative damage, such as: cerebral ischemia, diabetes mellitus, ischemic reperfusion, Parkinson's disease Parkinson's disease, shock, disseminated intravascular coagulation syndrome, atherosclerosis, neurodegenerative disease, arthritis, gastric ulcer Ulcers), Alzheimer's Disease, ulcerative colitis, cancer, Down's syndrome, macular degeneration (macular 12 201043239 degeneration), etc. The present invention also provides a pharmacy A composition comprising a peptide as described above as a primary anti-inflammatory agent. The pharmaceutical composition is contemplated for use in the treatment or prevention of inflammatory diseases such as stroke, multiple sclerosis, encephalitis, septic shock. Adult respiratory distress syndrome, myocarditis, arthritis, neuritis, and glomerulonephritis. Further, the present invention provides a pharmaceutical composition comprising a peptide as described above as an active ingredient for inhibiting the growth of cancer cells, particularly gastric adenocarcinoma cells. The peptides according to the present invention are also expected to be used in the preparation of health foods or over-the-counter medicines for antihypertensive, anti-oxidant, anti-inflammatory and anti-cancer applications. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be further described with respect to the following examples, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as The implementation of the restrictions. EXAMPLES Experimental Materials: 1. The chlorella waste (waste of C7i/ore//a vw/gari··?) was purchased from Douliu Algae Farm, which is in the manufacturing process of algae essence. Medium, 13 201043239 Waste obtained after one-water extraction treatment of chlorella (C/i/ore//iz vw/garb). Chlorella waste was dried by hot air before being subjected to the following experiment and stored at -20 ° C for use. 2_protease: flavourzyme type A and alcalase were purchased from Novo Nordisk A/S (Copenhagen, Denmark); papain was purchased from Amano (Nagoya, Japan); Pepsin was purchased from Nacalai Tesque (Kyoto, Japan); and pancreatin was purchased from Sigma Chemical Co_ (St. Louis, MO, USA).

3. 細胞：人類胃腺癌細胞株 AGS (human gastric adenocarcinoma cell line AGS)、人類正常肺細胞 WI-38 (human normal lung cell WI-38)以及小鼠巨嗔細胞（RAW 264.7)是購自財團法人食品工業發展研究所（新竹，台灣） 〇 4. 杜貝可氏改良的依格氏培養基（Dulbecco’s Modified Eagle Medium, DMEM)以及 Roswell Park Memorial Institute (RPMI)培養基是購自於 JRH Biosciences (Kansas, USA)。 5. 分析管柱：Sephacryl S-100 HR 管柱、Q-sepharose Fast Flow 管柱以及 Sephasil peptide C8 管柱是購自 Pharmacia Biotech. Co· (Uppsala, Sweden);以及 Inertsil ODS-3 C18 半製備級管柱是購自GL Sciences Inc. (Japan)。 6. 下列實驗材料是購自 Sigma Chemical Co. (St. Louis, MO, USA):馬尿醯-L-組胺酸_L-白胺酸（Hippuryl-L-histidyl-L-Leucine, HHL)、血管緊縮素 I-轉化酵素（angiotensin I- 14 201043239 converting enzyme, ACE)(得自人類皮膚）、馬尿酸 (hippuric acid, HA)、2,2’-次偶氮基雙（3-乙基苯並嚷唾琳-6-績酸）[2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)，ABTS]、1，1-二苯基-2-三硝基苯肼（l，l-diphenyl-2-picrylhydrazyl, DPPH)自由基、0弓丨經-β-D-葡萄糖搭酸 , (indoxyl-P-D-glucuronide, IBG)、FeS〇4 · 7H20、光澤精 (lucigenin)、精胺酸（arginine)、丙酮酸 (methylglyoxal)、 3-(4,5-二甲基-2-基）-2,5-二苯基四唑溴化物[3-(4,5-◎ dimethyl-2-yl)-2,5-diphenyltetrazolium bromide]、胎牛血 清（fetal bovine serum, FBS)，以及 3-(4,5-二曱基嗜0坐-2-基）-2,5-二苯基四嗤溴化物[3-(4,5-0丨〇^化丫1化丨32〇1-2->4)-2,5-diphenyltetrazolium bromide, MTT]。 7·其它實驗材料：乙二胺四乙酸（ethylenediaminetetraacetic acid, EDTA)以及 H2〇2 (30%，v/v)是購自 Merck Co_ (Darmstadt，German);榮光素（fluorescein)、6-經基-3,5,7,8-四曱基-2-羧酸（6-hydroxy-2,5,7,8-tetramethyl-2-〇 carboxylic acid)(Trolox)以及 2,2’-偶氮雙（2-甲脒基丙烧） 二氣化氫[2,2’-Azobis(2-amidinopropane)dihydrochloride， AAPH]是購自 Aldrich (Milwaukee，WI，USA);載體 pET-28a (+)是購自 Novagen (Madison, WI，USA);以及 SYBR Green 是購自 Roche (Indianapolis, USA)。 一般實驗方法： A、ACE 抑制活性的測量（Measurement of ACE inhibitory activity): 15 201043239 有關ACE抑制活性的測量是參照D.W_ Cushman and H.S. Cheung (1971), ·ΡΛα/·*7ηα<：0/·，20:1637-1648 當 中所述的方法並略作修改後來進行。簡言之，一由100 mM 的硼酸鈉緩衝液（pH 8.3)、1.68 mU的ACE以及15 μι的待 測樣品所形成之混合物（190 μί)於37°C下被預培育歷時5分 鐘，繼而加入15 μί的HHL至一最終濃度為3.94 mM，藉 此以起始反應。接著，在37°C下培育歷時1小時之後，加 入190 pL的1 M HC1來終止反應，藉此而得到一測試溶液 (test solution)。另外，對照組是以去離子水來取代待測樣品 〇 取 5 μί 的測試溶液來進行管柱層析（column chromatography)，其中所使用.的管柱以及操作條件如下： 分析管柱為 Inertsil [十八基矽烷（octadecylsilane)] ODS-3 C18管柱，長度：4.6 mm X 250 mm ;流動相：0.1%三氟乙 酸（trifluoroacetic acid, TFA)(配於50%甲醇中）；流速被控制 為0.8 mL/min ;偵測波長被設定為228 nm。 待測樣品的ACE抑制活性是藉由將所測得的吸光值 (OD228)代入下列公式而被計算出： A(%)= [(B-C)/B]xl00 其中：A=ACE抑制活性 B=對照組的OD228數值 C =測試溶液的0 D 2 2 8數值 B、Trolox當量抗氧化物能力分析丨Trolox equivalent antioxidant capacity (TEAC) assay】： 16 201043239 有關測量抗氧化能力的TEAC分析是參考M.J.T.J. Arts ei α/· (2004), Fooi/ CTiem·，88:567-57 當中所述的方法並略作 修改後來進行。簡言之，將10 μί的待測樣品與200 pL的 ABTS溶液充分混合以形成一混合物，於室溫下靜置2分鐘 之後，在一為734 nm的波長下測定吸光值（OD734)。另外， . 對照組（control)是以製備待測樣品時所使用的溶劑來取代待 測樣品。 待測樣品的ABTS自由基清除活性是藉由將所測得的吸 Ο 光值（〇d734)代入下列公式而被計算出： A(%)= [(B-C)/B]xl00 其中：A=ABTS自由基清除活性 B=對照組的OD734數值 C=混合物的OD734數值 C、統計學分析（Statistical analysis): 在下面的實施例中，各組的實驗被重複進行3次，而 ' 所得到的實驗數據是以“平均值土標準偏差（standard3. Cells: human gastric adenocarcinoma cell line AGS, human normal lung cell WI-38, and mouse giant cell (RAW 264.7) were purchased from the consortium. Food Industry Development Institute (Hsinchu, Taiwan) 〇 4. Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) medium were purchased from JRH Biosciences (Kansas, USA) ). 5. Analytical column: Sephacryl S-100 HR column, Q-sepharose Fast Flow column and Sephasil peptide C8 column are available from Pharmacia Biotech. Co. (Uppsala, Sweden); and Inertsil ODS-3 C18 semi-preparative grade The column was purchased from GL Sciences Inc. (Japan). 6. The following experimental materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA): Hippuryl-L-histidyl-L-Leucine (HHL) , angiotensin I- 14 (201043239 converting enzyme, ACE) (from human skin), hippuric acid (HA), 2,2'-azo bis (3-ethyl [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS], 1,1-diphenyl-2-trinitrophenylhydrazine (l, L-diphenyl-2-picrylhydrazyl, DPPH) free radicals, 0- 丨-β-D-glucose acid, (indoxyl-PD-glucuronide, IBG), FeS〇4 · 7H20, lucigenin, spermine Acid (arginine), pyruvate (methylglyoxal), 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide [3-(4,5-◎ dimethyl-2) -yl)-2,5-diphenyltetrazolium bromide], fetal bovine serum (FBS), and 3-(4,5-diindolyl-2-ylidene-2-yl)-2,5-diphenyl Tetrakilium bromide [3-(4,5-0丨〇^丫1丫丨32〇1-2->4)-2,5-diphenyltetrazolium bromide, MTT]. 7. Other experimental materials: ethylenediaminetetraacetic acid (EDTA) and H2〇2 (30%, v/v) were purchased from Merck Co_ (Darmstadt, German); fluorescein, 6-mer radical -3,5,7,8-tetramethyl-2-carboxylic acid (Trolox) and 2,2'-azo double (2-methylmercaptopropene) 2,2'-Azobis (2-amidinopropane) dihydrochloride, AAPH] was purchased from Aldrich (Milwaukee, WI, USA); the carrier pET-28a (+) was purchased. From Novagen (Madison, WI, USA); and SYBR Green is available from Roche (Indianapolis, USA). General Experimental Methods: A, Measurement of ACE inhibitory activity: 15 201043239 For the measurement of ACE inhibitory activity, refer to D.W_Cushman and HS Cheung (1971), ·ΡΛα/·*7ηα<:0/ ·, 20:1637-1648 The method described in the following was slightly modified. Briefly, a mixture of 100 mM sodium borate buffer (pH 8.3), 1.68 mU ACE, and 15 μm of the sample to be tested (190 μί) was pre-incubated at 37 ° C for 5 minutes, and then 15 μί of HHL was added to a final concentration of 3.94 mM to initiate the reaction. Next, after incubation at 37 ° C for 1 hour, 190 pL of 1 M HCl was added to terminate the reaction, thereby obtaining a test solution. In addition, the control group replaced the sample to be tested with deionized water and took 5 μί of the test solution for column chromatography. The column and operating conditions used were as follows: The analytical column was Inertsil [ Octadecylsilane ODS-3 C18 column, length: 4.6 mm X 250 mm; mobile phase: 0.1% trifluoroacetic acid (TFA) (in 50% methanol); flow rate is controlled to 0.8 mL/min; the detection wavelength was set to 228 nm. The ACE inhibitory activity of the sample to be tested is calculated by substituting the measured absorbance value (OD228) into the following formula: A (%) = [(BC) / B] xl00 where: A = ACE inhibitory activity B = OD228 value of the control group C = 0 D 2 2 of the test solution. Value B, Trolox equivalent antioxidant capacity analysis (TEAC) assay: 16 201043239 TEAC analysis for measuring antioxidant capacity is referred to MJTJ Arts Ei α/· (2004), Fooi/CTiem·, 88:567-57 The method described in the following was carried out with minor modifications. Briefly, 10 μί of the sample to be tested was thoroughly mixed with 200 pL of ABTS solution to form a mixture, and after standing at room temperature for 2 minutes, the absorbance (OD734) was measured at a wavelength of 734 nm. In addition, the control is a substitute for the sample to be tested in the preparation of the sample to be tested. The ABTS radical scavenging activity of the sample to be tested is calculated by substituting the measured sucking light value (〇d734) into the following formula: A(%) = [(BC)/B]xl00 where: A= ABTS free radical scavenging activity B = OD734 value of the control group C = OD734 value of the mixture C, statistical analysis: In the following examples, the experiments of each group were repeated 3 times, and the obtained The experimental data is based on the mean mean standard deviation (standard

Ci deviation, S.D.)”來表示。所有實驗數據是以史徒登氏ί-試 驗（Student’s /-test)來評估實驗組與對照組之間的差異性。 若所得到的統計分析結果是P < 0.05，這表示有統計顯著性 (statistical significance)。 實施例1.從小球藻廢棄物的蛋白晦水解物中篩選出具有 ACE抑制活性的肽 為了從小球藻廢棄物中獲得具有ACE抑制活性的肽， 申請人分別使用4種市售的蛋白酶（亦即A型風味蛋白酶、 17 201043239 驗2梅、木瓜蛋白酶以及胃蛋白酶）來水解小球藻廢棄物 ’並且猎此所得到的水解物（hydn)lysatem拿來進行蛋白質 產率（protein yield)以及ACE抑制活性（ace i祕i如 activity)的分析。 實驗方法： A、小球藻廢棄物的蛋白晦水解物之製傷： 小球藻廢棄物被分成4個實驗組（亦即風味蛋白梅組、 鹼蛋白酶組、木瓜蛋白酶組以及胃蛋白酶組)，各實驗組是 依照下面的操作步驟以及表i中所述的操作條件來進行水 解反應。 表1·各實驗組用於進行水解反應的操作條件 組別 蛋白酶 pH值 溫度（°C) 風味蛋白酶組 A型風味蛋白酶 6.0 50 驗蛋白酶組 驗蛋白酶 7.5 木瓜蛋白梅組 木瓜蛋白酶 7.5 胃蛋白酶組 胃蛋白酶· 2.0 首先’將10 g的小球藻廢棄物配於100 mL的去離子水 中以形成一具有一濃度為10% (w/v)的小球藻廢棄物溶液。 將該溶液的pH值調整至預定的pH值，繼而加入適量的蛋 白酶至一最終濃度為〇_2% (w/v)，藉此而形成一反應混合物 。所形成的反應混合物被置於50°C下進行水解歷時15小時 ’之後’將之移至一沸水浴（boiling water bath)中並且予以 加熱歷時10分鐘，藉此將蛋白酶去活化（inactivate)。接著 ，在10,000 rpm下予以離心歷時20分鐘，之後，收取上清 18 201043239 液進行下面的分析。 B、 總蛋白質含量的測定： 有關各實驗組的總蛋白質含量的測定是藉由使用微 BCA 蛋白質分析套組（micro BCA protein assay kit)(Bio-Rad， USA)並依照廠商所提供的操作指引來進行。牛血清白蛋白 . (bovine serum albumin)被使用作為標準品。 另外，在本實驗中是以未經蛋白酶水解的小球藻廢棄 物作為對照組，小球藻廢棄物的總蛋白質含量是藉由使用 〇 凱氏氮素分析（Kjeldahl nitrogen determination)被計算出， 而有關凱氏氮素分析的操作步驟可參見，例如，食品工業 發展研究所編印的食品分析方法手冊（1990)。 上面各組所測得的總蛋白質含量被代入下列公式中以 計算出各組的蛋白質產率： A(%)=(B/C)xl00 其中：A=蛋白質產率 — B=實驗組的總蛋白質含量 Ο Ο對照組的總蛋白質含量 C、 ACE抑制活性的測量： 在上面A項當中所得到的上清液是依照上面“一般實驗 方法”的“A、ACE抑制活性的測量”當中所述方法來測定 ACE抑制活性。 於上面各項分析中所得到的實驗數據是依照上面“一般 實驗方法”的“C、統計學分析”當中所述方法來進行分析。 .结采.· 19 201043239 圖1A與圖1B分別顯示藉由使用不同的蛋白酶來對小 球藻廢棄物進行水解所得到的水解物的蛋白質產率以及 ACE抑制活性。從圖1A與圖1B可見，各實驗組的蛋白質 產率以及ACE抑制活性與對照組所具者相較均呈現出統計 顯著性b<0.05)，其中以胃蛋白酶組顯示出最高的ACE抑 制活性以及蛋白質產率。因此，在下面的實驗中，申請人 將從小球藻廢棄物的胃蛋白酶水解物中進一步分離出具有 ACE抑制活性的肽。 實施例2.具有ACE抑制活性的肽的分離舆鑑定： 實驗方法： A、硫酸按分級分離處理（ammonium sulfate fractionation) 將適量的硫酸銨缓慢加入至上面實施例1中所得到的 小球藻廢棄物的胃蛋白酶水解物中，直到所形成的溶液具 有一為20%的硫酸銨飽和度。接著，在10,000 g下離心歷 時20分鐘而得到一上清液以及一沉澱物。該沉澱物被散浮 於10 mL的去離子水中，藉此而得到對應於0〜20%的硫酸 銨飽和度的分離部分A1。 另外，將所得到的上清液重複進行上述實驗並依次調 整硫酸胺飽和度至40%、60%以及80%，藉此而依序得到對 應於20〜40%、40〜60%以及60〜80%的硫酸銨飽和度的分離 部分A2、A3與A4。 之後，各分離部分分別被倒入一具有一為1,000道耳頓 (dalton)的分子量截斷值（molecular weight cut-off value)的透 20 201043239 析膜（dialysis membrane)(CelluSep)中並置於去離子水中進行 透析。經透析的各分離部分是依照上面“一般實驗方法”的 “A、ACE抑制活性的測量”當中所述方法來測定ACE抑制 活性。所得結果被顯示於圖2中。 從圖2可見，分離部分A3與A4的ACE抑制活性相較 . 於分離部分A1或A2所具者是較高的。因此，申請人將分 離部分A3與A4混合並予以冷凍乾燥，所得到的混合物被 用來進行下面的分析。 〇 B、凝耀過遽廣析（gel filtration chromatography): 將上面A項當中所得到的分離部分A3與A4的混合物 溶於5 mL的去離子水中，並依據下面的操作條件來進行凝 膠過濾層析：分析管柱為一經去離子水預平衡（pre- equilibrated)的 Sephacryl S-100 high HR 管柱（長度：2.6 cm χ 70 cm);洗提液為去離子水；流速被控制為1.5 mL/min ; 偵測波長被設定為215 nm。於洗提時，將每6 mL的洗出物 ' (eluate)收集於一個試管内。 Ο 依據所得到的洗提圖形（elution profile)來收集各波峰所 對應的洗出物’繼而依照上面“一般實驗方法”的“A、ACE 抑制活性的測量”當中所述方法來測定ACE抑制活性。 實驗結果發現：分離部分A3與A4的混合物藉由使用 凝膠過濾層析而分離出兩個具有ACE抑制活性的分離部分（ 亦即分離部分B1與B2)。分離部分B2的ACE抑制活性相 較於分離部分B1所具者是較高的（參見圖3)。因此，將分 離部分B2冷凍乾燥以供進行下面的分析。 21 201043239 C、 陰離子交換廣析（anion exchange chromatography): 將上面B項當中所得到的經冷凍乾燥之分離部分B2溶 於5 mL的去離子水中，並依據下面的操作條件來進行陰離 子交換層析：分析管柱為一經20 mM Tris-HCl緩衝溶液（pH 7.8)預平衡的 Q-sepharose Fast Flow 管柱（長度：2.6 cm X 40 cm);洗提液為具有一 NaCl梯度（0.0至1.0 Μ)的20 mM Tris-HCl緩衝溶液(pH 7.8);流速被控制為1.5 mL/min ;偵 測波長被設定為215 nm。於洗提時，將每6 mL的洗出物收 集於一個試管内。 f 依據所得到的洗提圖形來收集各波峰所對應的洗出物 ，繼而依照上面“一般實驗方法”的“A、ACE抑制活性的測 量”當中所述方法來測定ACE抑制活性。 實驗結果發現：當洗提液的NaCl濃度為0 Μ以及 NaCl濃度約介於0.35 Μ和0.45 Μ之間時，分別有1個波 峰被觀察到。收集該等波峰所對應的洗出物而分別得到分 離部分C1與C2 (參見圖4Α)，其中分離部分C1具有強的 ACE抑制活性，而分離部分C2不具有ACE抑制活性（參見 ！ 圖4B)。因此，將分離部分Cl冷凍乾燥以供進行下面的分 析。 D、 逆相高效能液相層析（reverse phase-high performance liquid chromatography, RP-HPLC) · 將上面C項當中所得到的經冷凍乾燥之分離部分Cl溶 於1 mL的去離子水中，並依據下面的操作條件來進行逆相 高效能液相層析：儀器型號為 Agilent 1100 (Agilent 22 201043239Ci deviation, SD)". All experimental data were based on the Student's /-test to assess the difference between the experimental group and the control group. If the statistical analysis obtained is P &lt 0.05, which means statistical significance. Example 1. Screening of peptides with ACE inhibitory activity from peptone hydrolysate of chlorella waste in order to obtain ACE inhibitory activity from chlorella waste For peptides, the applicant used four commercially available proteases (ie, type A flavor protease, 17 201043239 to test 2 plum, papain, and pepsin) to hydrolyze chlorella waste' and to obtain the hydrolyzate obtained therefrom (hydn Lysatem is used for the analysis of protein yield and ACE inhibitory activity (ace i) such as activity. Experimental method: A. Damage to peptone hydrolysate of chlorella waste: chlorella abandonment The material was divided into 4 experimental groups (ie, flavor protein plum group, alkali protease group, papain group and pepsin group), each experimental group was in accordance with the following steps and in Table i The operating conditions are used to carry out the hydrolysis reaction. Table 1. The operating conditions of each experimental group for the hydrolysis reaction, the protease pH temperature (°C), the flavor protease group, the type A flavor protease, 6.0, the protease group, the protease 7.5, the papaya Protein plum group papain 7.5 Pepsin group pepsin · 2.0 First '10 g of chlorella waste was mixed in 100 mL of deionized water to form a 10% (w/v) chlorella Waste solution. The pH of the solution is adjusted to a predetermined pH, and then an appropriate amount of protease is added to a final concentration of 〇 2% (w/v), thereby forming a reaction mixture. The hydrolysis was carried out at 50 ° C for 15 hours 'after' and was transferred to a boiling water bath and heated for 10 minutes, whereby the protease was inactivated. Then, at 10,000 rpm Centrifuge for 20 minutes, then collect the supernatant 18 201043239 for the following analysis. B. Determination of total protein content: Measurement of total protein content of each experimental group It was carried out by using the micro BCA protein assay kit (Bio-Rad, USA) according to the manufacturer's instructions. Bovine serum albumin was used as a standard. In addition, in this experiment, chlorella waste without protease hydrolysis was used as a control group, and the total protein content of chlorella waste was calculated by using Kjeldahl nitrogen determination. For the procedure for Kjeldahl nitrogen analysis, see, for example, the Handbook of Food Analysis Methods (1990), published by the Food Industry Development Institute. The total protein content measured in each of the above groups was substituted into the following formula to calculate the protein yield of each group: A (%) = (B / C) xl00 where: A = protein yield - B = total of the experimental group Protein content Ο 总 Total protein content of the control group C. Measurement of ACE inhibitory activity: The supernatant obtained in the above item A is as described in "Measurement of A, ACE inhibitory activity" according to the "general experimental method" above. Methods to determine ACE inhibitory activity. The experimental data obtained in the above analyses were analyzed in accordance with the method described in "C, Statistical Analysis" of the "General Experimental Method" above. .. 19 201043239 FIG. 1A and FIG. 1B respectively show the protein yield and ACE inhibitory activity of the hydrolyzate obtained by hydrolyzing chlorella waste using different proteases. As can be seen from Fig. 1A and Fig. 1B, the protein yield and ACE inhibitory activity of each experimental group showed statistical significance b<0.05) compared with those of the control group, wherein the highest ACE inhibitory activity was exhibited by the pepsin group. And protein yield. Therefore, in the following experiment, the applicant further separates the peptide having ACE inhibitory activity from the pepsin hydrolysate of the chlorella waste. Example 2. Isolation of peptides having ACE inhibitory activity 舆 Identification: Experimental method: A. Sulfuric acid fractionation treatment (ammonium sulfate fractionation) An appropriate amount of ammonium sulfate was slowly added to the chlorella obtained in the above Example 1 to be discarded. The pepsin hydrolysate of the product until the solution formed has a saturation of 20% ammonium sulfate. Then, it was centrifuged at 10,000 g for 20 minutes to obtain a supernatant and a precipitate. The precipitate was dispersed in 10 mL of deionized water, whereby a separated portion A1 corresponding to 0 to 20% of ammonium sulfate saturation was obtained. Further, the obtained supernatant was repeatedly subjected to the above experiment and the ammonium sulfate saturation was sequentially adjusted to 40%, 60%, and 80%, whereby sequentially obtained corresponding to 20 to 40%, 40 to 60%, and 60 to Separated portions A2, A3 and A4 of 80% ammonium sulfate saturation. Thereafter, each of the separated fractions was respectively poured into a dialysis membrane (CelluSep) having a molecular weight cut-off value of 1,000 daltons and placed in a deionized ion. Dialysis in water. Each of the separated fractions subjected to dialysis was assayed for ACE inhibitory activity according to the method described in "Measurement of A, ACE inhibitory activity" of "General Experimental Method" above. The results obtained are shown in Figure 2. As can be seen from Fig. 2, the separation portion A3 is compared with the ACE inhibitory activity of A4. It is higher in the separation portion A1 or A2. Therefore, the applicant mixed the separated portions A3 and A4 and freeze-dried, and the resulting mixture was used for the following analysis. filtrationB, gel filtration chromatography: The mixture of the separated fractions A3 and A4 obtained in the above item A is dissolved in 5 mL of deionized water, and subjected to gel filtration according to the following operating conditions. Chromatography: The analytical column was a pre-equlibrated Sephacryl S-100 high HR column (length: 2.6 cm χ 70 cm); the eluent was deionized water; the flow rate was controlled to 1.5. mL/min ; The detection wavelength is set to 215 nm. At the time of elution, each 6 mL of eluate was collected in a test tube.收集 Collect the eluate corresponding to each peak according to the obtained elution profile'. Then determine the ACE inhibitory activity according to the method described in “Measurement of A, ACE Inhibitory Activity” in “General Experimental Methods” above. . As a result of the experiment, it was found that the separated fraction of A3 and A4 was separated into two separated fractions having ACE inhibitory activity (i.e., separated fractions B1 and B2) by using gel filtration chromatography. The ACE inhibitory activity of the fraction B2 was higher than that of the fraction B1 (see Fig. 3). Therefore, the separated portion B2 was freeze-dried for the following analysis. 21 201043239 C. Anion exchange chromatography: The lyophilized fraction B2 obtained in the above item B is dissolved in 5 mL of deionized water and subjected to anion exchange chromatography according to the following operating conditions. : Analytical column is a Q-sepharose Fast Flow column (length: 2.6 cm X 40 cm) pre-equilibrated with 20 mM Tris-HCl buffer solution (pH 7.8); the eluent has a NaCl gradient (0.0 to 1.0 Μ) 20 mM Tris-HCl buffer solution (pH 7.8); flow rate was controlled to 1.5 mL/min; detection wavelength was set to 215 nm. At the time of elution, each 6 mL of the eluate was collected in one test tube. f The eluate corresponding to each peak was collected based on the obtained elution pattern, and then the ACE inhibitory activity was measured in accordance with the method described in "Measurement of A, ACE inhibitory activity" of "General Experimental Method" above. The experimental results show that when the NaCl concentration of the eluent is 0 Μ and the NaCl concentration is between 0.35 Μ and 0.45 ,, one peak is observed. The eluates corresponding to the peaks were collected to obtain separate fractions C1 and C2, respectively (see Fig. 4A), wherein the fraction C1 had strong ACE inhibitory activity, while the fraction C2 did not have ACE inhibitory activity (see Fig. 4B). . Therefore, the separated portion C1 was freeze-dried for the following analysis. D. reverse phase-high performance liquid chromatography (RP-HPLC) · Dissolve the lyophilized fraction C obtained in item C above in 1 mL of deionized water, and The following operating conditions were used for reverse phase high performance liquid chromatography: the instrument model was Agilent 1100 (Agilent 22 201043239)

Technologies Inc. CA，USA);分析管柱為 inertsH 〇Ds_3 C18半製備級管柱（10 mm x 250 mm);洗提液為具有一乙 腈梯度（acetonitrile gradient)(25 至 40%，30 分鐘）的含有 0 _ 1 % TFA之水溶液，流速被控制為2 mL/min ;偵測波長被 設定為215 nm。 - 依據所得到的洗提圖形來收集各波峰所對應的洗出物 . ，繼而依照上面“一般實驗方法”的“A、ACE抑制活性的測 量”當中所述方法來測定ACE抑制活性。 實驗結果發現.在滯留時間約為16至17分鐘時有一 明顯的波峰被觀察到（參見圖5 A)，並且該波峰所對應的洗 出物（亦即分離部分D1)具有高的ACE抑制活性。 將分離部分D1冷凍乾燥並溶於〇丨mL的去離子水中 ，繼而依據下面的操作條件再進行一次逆相高效能液相層 析：分析管柱為8叩1^丨1?6州(^〇8管柱(4.6父25〇111111); 洗提液為具有一乙腈梯度（acetonitrile gradient)(25至4〇%， 30分鐘）的含有〇1% TFA之水溶液；流速被控制為J 0 mL/min ;偵測波長被設定為215 nm。 依據所得到的洗提圖形來收集各波峰所對應的洗出物 ，繼而依照上面“ 一般實驗方法，，的“A、ACE抑制活性的測 量”當中所述方法來測定ACE抑制活性。 實驗結果發現：在滞留時間約為14至15分鐘時偵測 到明顯的波峰（參見圖5B)，並且該波峰所對應的洗出物（ 亦P刀離分P1)具有ACE抑制活性。將分離部分Η冷凍 乾燥而得到具有ACE抑制活性的肽。 23 201043239 E、胺基酸序列分析： 在上面D項當中所得到的肽是藉由使用 Applied Biosystems Procise 494 蛋白質定序儀（Applied Biosystems Procise 494 protein sequencer, Foster City, CA，USA)並參照 廠商所提供的操作指引來進行胺基酸序列分析。 分析結果顯示：所得到的肽具有如下所示之胺基酸序 列：Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe (序列 辨識編號：1)，並且具有一分子量為1，309 Da。 另外，申請人為了驗證在本實施例中所得到的肽確實 具有ACE抑制活性，一具有如序列辨識編號：1所示的胺 基酸序列的肽被合成出，並且依照上面“一般實驗方法”的 “A、ACE抑制活性的測量”當中所述方法來測定ACE抑制 活性。 結果發現：合成的肽亦具有如同在本實施例中所得到 的肽的ACE抑制活性（數據未顯示）。這個結果證實：在本 實施例中所分離得到的具有序列辨識編號：1所示之胺基酸 序列的肽確實具有ACE抑制活性。 實施例3.分離自小球藻廢棄物的胃蛋白梅水解物的肽在 ACE抑制特性上的分析 為瞭解依據本發明之分離自小球藻廢棄物的胃蛋白酶 水解物的肽對於ACE的抑制特性，申請人使用實施例2當 中所得到的具有ACE抑制活性的肽來進行下面的實驗。 實驗方法： A、肽的濃度對於ACE抑制活性的影響： 24 201043239 將適量的從上面實施例2中所得到的肽溶於去離子水 中以形成濃度分別為8.78、17.55、21.61以及43.23 μΜ的 肽溶液（peptide solution)，另外，去離子水被用來作為對照 組。各個濃度的肽溶液是依照上面“一般實驗方法”的“A、 ACE抑制活性的測量”當中所述方法來測定ACE抑制活性 ，並且計算出肽的抑制50% ACE活性的濃度（IC5Q)。所得到 的實驗數據是依照上面“一般實驗方法”的“C、統計學分析” 當中所述方法來進行分析。 B、肽對於 ACE 的抑制態樣（inhibition pattern): 將適量的從上面實施例2中所得到的肽溶於去離子水 中以形成濃度分別為0.02以及0.04 mM的肽溶液，另外， 去離子水被用來作為對照組。 各個濃度的肽溶液大體上是依照上面“一般實驗方法”的 “A、ACE抑制活性的測量”當中所述方法來測定ACE抑制 活性，不同之處在於：實驗被重複進行4次，並且在各次 的測定中所使用的HHL濃度分別為3.94、1.97、0.985以及 0.493 mM。由此所得到的實驗結果被繪成Lineweaver-Burk 圖（Lineweaver-Burkplots)來進行分析。 · A、肽的濃度對於ACE抑制活性的影響： 圖6顯示分離自小球藻廢棄物的胃蛋白酶水解物的肽 在不同濃度下的ACE抑制活性。從圖6可見，ACE抑制活 性會隨著肽濃度的增加而升高，而所計算出的IC5〇數值是 29.6 μΜ。這個結果顯示：分離自小球藻廢棄物的胃蛋白酶 25 201043239 水解物的狀對於ACE具有一劑量-依賴性抑制作用。 B、肽對於ACE的抑制態樣： 圖7顯示分離自小球藻廢棄物的胃蛋白酶水解物的肽 在不同濃度下對於ACE的抑制活性的Lineweaver-Burk圖 。從圖7可見，Lineweaver-Burk圖的斜率以及與Y軸相交 的截距會隨著肽濃度的增加而升高，但與X軸相交的截距 卻沒有改變。因此，分離自小球藻廢棄物的胃蛋白酶水解 物的肽對於ACE的抑制模式（inhibition mode)是屬於非競爭 型抑制（non-competitive inhibition)，這表示：無論是否存在 有ACE的受質，分離自小球藻廢棄物的胃蛋白酶水解物的 肽均可結合至ACE。申請人推測：分離自小球藻廢棄物的 胃蛋白酶水解物的肽可能會造成ACE的構形改變 (conformational change)進而防止ACE將受質轉化成產物， 因此無論是存在有低濃度或者高濃度的受質，該肽均可達 到相同的ACE抑制效用。 實施例4.溢度、pH值以及胃腸道蛋白ft|(gastrointestinal protease)對於分離自小球藻廢棄物的胃蛋白梅水 解物的肽的影響 為進一步探討溫度、pH值以及胃腸道蛋白酶對於依據 本發明之分離自小球藻廢棄物的胃蛋白酶水解物的肽在 ACE抑制效用上的影響，申請人使用實施例2當中所得到 的具有ACE抑制活性的肽來進行下面的實驗。 實驗方法： A、溫度對於肽的影響： 26 201043239 將上面實施例2中所得到的肽溶於去離子水中以形成 一具有一濃度為1%的肽溶液。將所得到的肽溶液分成4組 並分別於40、60、80以及10(TC下靜置歷時！小時，繼而 依照上面“ 一般實驗方法，’的“ A、A C E抑制活性的測量，，當中 所述方法來測定各組肽溶液的ACE抑制活性。 B、pH值對於肽的影響： 將上面實施例2中所得到的肽溶於去離子水中以形成 一具有一濃度為1%的肽溶液。將所得到的肽溶液分成5組 〇 並將pH值分別調整至2、4、6、8以及10，繼而於37t下 靜置歷時1小時。之後，依照上面“一般實驗方法，，的“A、 ACE抑制活性的測量”當中所述方法來測定各組肽溶液的 ACE抑制活性。 c、胃腸道蛋白晦對於肽的影響： 有關胃腸道蛋白酶對於肽的影響主要是參照j Wu an(j X· Ding (2QQ2\ Food Research International, 35:36Ί-375 當 中所述的方法並略作修改後來進行評估。簡言之，將1 g的 ϋ 從上面實施例2中所得到的肽溶於10 mL的0.1 M KC1-HC1 緩衝溶液（pH 2.0)中，繼而加入o.oi g的胃蛋白酶，藉此以 形成一混合物。將所形成的混合物置於一具有一溫度為37 C的水浴槽中進行培育歷時3小時。之後，將該混合物的 pH值調整至7.8，繼而予以加熱至沸騰歷時1〇分鐘，俾以 將胃蛋白酶去活化。 待3亥混合物冷卻之後，加入〇.〇1 g的胰酶（pancreatin) ’繼而置於一具有一溫度為37°C的水浴槽中進行培育歷時 27 201043239 4小時。之後’予以加熱至沸騰歷時ι〇分鐘，接而在 1〇，_ g下予以離心歷時10分鐘。所得到的上清液是依照 上面“一般實驗方法，，的“A、ACE抑制活性的測量，，當中所述 方法來測定ACE抑制活性。 結果： 有關PH值與溫度對於分離自小球藻廢棄物的胃蛋白酶 水解物的肽的影響，由實驗結果發現：本發明的肽在分別 置於不_PH值(PH2、4、6、8以及1〇)以及不同的溫度 ⑽^冬以及戰邝進行培育歷日”小時之後仍然 具有ACE抑制活性（數據未顯示）。申請人據此而推論：本 發明的肽具有優越的pH-以及熱_安定性（pH_ and “仏 stability)。 另外，有關胃腸道蛋白崎對於本發明的肽的影響，由 實驗結果發現：在活體外依序地與胃蛋白酶以及胰酶進行 培育之後’本發明的肽# ACE抑制活性與對照组所具者相 較並無呈現出統計顯著性⑺>0.05)(數據未顯示）。申請人據 此而推論：本發明的肽對於胃腸道酵素消化作用 (gastrointestinal enZyme digesti〇n)具有抗性，因此本發明的 肽在攝食之後能夠克服人類消化道的環境壓力並發揮所欲 的效用。 從上述實驗結果可知，分離自小球藻廢棄物的胃蛋白 酶水解物的肽具有優異的ACE抑制活性以及良好的pH_與 熱-安定性，因而被預期可供用於製備一用來調節血壓的醫 藥品（medicaments)或膳食補充品（dietary supplements)。 28 201043239 實施例5.分離自小球藻廢棄物的胃蛋白酶水解物的肽對於 人類胃腺癌細跑株 AGS (human gastric adenocarcinoma cell line AGS)的生長抑制效用的 評估 為瞭解依據本發明之分離自小球藻廢棄物的胃蛋白酶 . 水解物的肽對於人類胃腺癌細胞株AGS的增生與分化有何 影響，申請人使用實施例2當中所得到的具有ACE抑制活 性的肽來進行下面的實驗。 〇 it驗方法· 下面有關抗增生效用的評估是參照S. Katayama and Y. Mine (2007)，·/· Jgr/c. Food 55:8458-8464 當中所述 的方法並略作修改後來進行。 首先，將人類胃腺癌細胞AGS接種至含有RPMI培養 基（含有10%胎牛血清）的96-井培養盤（96-well plate)(4xl03 細胞/井）内，並且在37°C下予以培養歷時24小時。之後， _ 加入適量的肽溶液（將上面實施例2中所得到的肽溶於去離 Ο 子水中）至一最終濃度分別為206、244以及306 μΜ，繼而 予以培養歷時72小時。另外，添加去離子水的細胞被用來 作為對照組。接著，將ΜΤΤ (20 μί/井）加入至各井内並進 行培養歷時4小時。之後，將200 μΐ之100%的二甲亞颯 (DMSO)添加至各井中，俾以溶解出ΜΤΤ-甲臢（ΜΤΤ-formazan)。於570 nm的波長下以一微量滴定盤讀取儀 (microplate reader)來讀取各井的吸光值（〇D57Q)。細胞生長 抑制率是藉由將所測得的OD57〇數值代入下面的公式而被計 29 201043239 算出： A(%)= [(B-C)/B]xl00 其中：A=細胞生長抑制率 B=對照組的OD57〇數值 Ο實驗組的OD57〇數值 所得到的實驗數據是依照上面“一般實驗方法”的“C、 統計學分析”當中所述方法來進行分析。 另外，申請人合成3個肽，亦即PEP1 (序列辨識編號 :2)、PEP2 (序列辨識編號：3)以及PEP3 (序列辨識編號： 4)來進行相同的實驗，這3個肽分別具有一胺基酸序列相同 於本發明的肽（序列辨識編號：1)的胺基酸序列之一部分（參 見表2)。 表2.具有一胺基酸序列相同於本發明的肽的胺基酸序列之 一部分的合成肽 肽名稱 胺基酸序列 ΡΕΡ1 Pro-Asn-Arg-Pro-Gln-Phe(序列辨識編號：2) ΡΕΡ2 Val-Glu-Cys-Tyr-Gly-Pro(序列辨識編號：3) ΡΕΡ3 匚>^-丁71'-01丫-?1'〇-八811-八^(序列辨識編號：4) #采·· 圖8顯示人類胃腺癌細胞AGS經不同濃度的肽處理之 後的細胞生長抑制率。從圖8可見，AGS細胞的細胞生長 抑制率與肽的濃度成正向關係，而所計算出的IC5Q數值為 256.4±1.2 μΜ。這個結果顯示：本發明的肽對於人類胃腺癌 細胞AGS的生長具有一劑量-依賴性抑制效用。 30 201043239 另外，與合成肽PEPl、PEP2以及PEP3相較之下，本 發明的肽展現出較優越的人類胃腺癌細胞AGS的生長抑制 效用（數據未顯示）。這個結果顯示：本發明的肽的序列以及 結構在針對人類胃腺癌細胞AGS的生長抑制效用上可能扮 演一重要的角色。 實施例6.分離自小球藻廢棄物的胃蛋白晦水解物的肽對於 誘導性 NO 生成(inducible NO production)的抑制 效用的評估 〇 實驗方法’· 下面有關誘導性NO生成的抑制效用是參照X. Yang W a/. (2008)，Food CAe/w·, 106:269-276 當中所述的方法並略作 修改後來進行評估。 首先，將小鼠巨噬細胞RAW 264.7接種至含有RPMI 培養基（含有10%的胎牛血清）的96-井培養盤（5xl04細胞/井） 内，並且在37°C下進行培養歷時4小時。之後，加入20 pL 的LPS (50 pg/mL)，並加入適量的肽溶液（將上面實施例2 〇 中所得到的肽溶於去離子水中）至一最終濃度分別為9.9、 19.9以及99.3 μΜ。另外，未添加肽溶液的細胞被用來作為 對照組。接著，在37°C下進行培養歷時24小時之後，於室 溫下取80 μί的上層液與160 μι的Griess試劑（Griess reagent)[l%的胺苯續醯胺（sulfanilamide)、0.1%的 N-萘基乙 烯二胺二氯化氫（N-naphthylethylenediamine dihydrochloride) 以及2.5%的磷酸]相混合並進行反應歷時20分鐘。以 ELISA 讀取儀（p〇werwave xs，Biotek，Vermont, USA)來測量 31 201043239 在波長540 nm下的吸光值（ODho)，將所獲得的〇d54()數值 與亞硝酸鈉（NaN〇2)的定量標準曲線比對而計算出亞硝酸鹽 產物（nitrite product)的濃度。NO生成抑制率是藉由將所測 得的亞硝酸鹽產物的濃度代入下列公式而被計算出： A(%)=[(B-C)/B]xl00 其中：A=NO生成抑制率 B=對照組的亞硝酸鹽產物的濃度 C=實驗組的亞硝酸鹽產物的濃度 潜求·· 圖9顯示小氣巨嘻細胞raw 264.7經不同濃度的肽處 理之後所測得的NO生成抑制率。從圖9可見，小鼠巨噬細 胞RAW 264.7的NO生成抑制率會隨著肽濃度的增加而升 高，而所計算出的ICS0數值是42·4 μΜ。這個結果顯示：分 離自小球藻廢棄物的胃蛋白酶水解物的肽對於誘導性Ν〇生 成具有一劑量-依賴性抑制作用。 從本實驗的結果可知：本發明的分離自小球藻廢棄物 的胃蛋白酶水解物具有抑制Ν〇生成的效用。由於Ν〇的生 成與發炎症狀的產生有關。因此，本發明的月太亦被預期具 有抗發炎的效用而可供用於製備一用來治療或預防發炎性 疾病的醫藥品或膳食補充品。 實施例7.具有抗氧化活性的肽的分離舆鑑定 在申請人的先前研究中曾發現小球藻廢棄物的胃蛋白 酶水解物具有抗氧化活性。因 U此在本實施例中，申請人 小球藻廢棄物的胃蛋白崎水解物中分離具有抗氧化 32 201043239 活性的狀。 實驗方法： A、小球藻廢棄物的胃蛋白晦水解物的製備： 將10 g的小球藻廢棄物配於100 mL的去離子水中以形 成一具有一濃度為10% (w/v)的小球藻廢棄物溶液。將該溶Technologies Inc. CA, USA); analytical column is inersH 〇Ds_3 C18 semi-preparative column (10 mm x 250 mm); eluent has an acetonitrile gradient (25 to 40%, 30 minutes) The aqueous solution containing 0 _ 1 % TFA, the flow rate was controlled to 2 mL / min; the detection wavelength was set to 215 nm. - The eluate corresponding to each peak was collected based on the obtained elution pattern, and then the ACE inhibitory activity was measured in accordance with the method described in "Measurement of A, ACE inhibitory activity" of "General Experimental Method" above. As a result of the experiment, it was found that a significant peak was observed when the residence time was about 16 to 17 minutes (see Fig. 5A), and the eluted product corresponding to the peak (i.e., the separated portion D1) had high ACE inhibitory activity. . The separated fraction D1 was freeze-dried and dissolved in deionized water of 〇丨mL, and then subjected to reverse phase high performance liquid chromatography according to the following operating conditions: the analytical column was 8叩1^丨1?6 (^ 〇8 column (4.6 parent 25〇111111); the eluent is an aqueous solution containing 〇1% TFA with an acetonitrile gradient (25 to 4%, 30 minutes); the flow rate is controlled to J 0 mL /min ; The detection wavelength is set to 215 nm. The eluate corresponding to each peak is collected according to the obtained elution pattern, and then according to the above "General Experimental Method, "Measurement of A, ACE Inhibitory Activity" The method was used to determine the ACE inhibitory activity. The experimental results showed that a distinct peak was detected when the residence time was about 14 to 15 minutes (see Fig. 5B), and the wash corresponding to the peak (also P knife split) P1) has ACE inhibitory activity. The isolated fraction of hydrazine is freeze-dried to obtain a peptide having ACE inhibitory activity. 23 201043239 E. Amino acid sequence analysis: The peptide obtained in the above item D is obtained by using Applied Biosystems Procise 494 protein Sequencer (Applied Bio The system Procise 494 protein sequencer, Foster City, CA, USA) was performed with reference to the manufacturer's instructions for amino acid sequence analysis. The results showed that the peptide obtained had the amino acid sequence shown below: Val-Glu -Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe (SEQ ID NO: 1), and has a molecular weight of 1,309 Da. In addition, the applicant has verified the results obtained in this example. The peptide does have ACE inhibitory activity, and a peptide having an amino acid sequence as shown in SEQ ID NO: 1 is synthesized, and the method described in "Measurement of A, ACE Inhibitory Activity" according to the "General Experimental Method" above The ACE inhibitory activity was measured. As a result, it was found that the synthesized peptide also had the ACE inhibitory activity as the peptide obtained in the present example (data not shown). This result confirmed that the sequence was isolated in the present example. The peptide of the amino acid sequence numbered 1: has indeed an ACE inhibitory activity. Example 3. Analysis of peptides of gastric protein plum hydrolysate isolated from chlorella waste in ACE inhibition characteristics for understanding According to the peptide of the pepsin hydrolysate isolated from chlorella waste according to the present invention, the following experiment was carried out by using the peptide having ACE inhibitory activity obtained in Example 2 for the inhibitory property of ACE. Experimental method: A Effect of peptide concentration on ACE inhibitory activity: 24 201043239 An appropriate amount of the peptide obtained in Example 2 above was dissolved in deionized water to form peptide solutions having concentrations of 8.78, 17.55, 21.61 and 43.23 μM, respectively. In addition, deionized water was used as a control group. Each concentration of the peptide solution was determined by the method described in "Measurement of A, ACE inhibitory activity" of "General Experimental Method" above, and the concentration of the peptide inhibiting 50% of ACE activity (IC5Q) was calculated. The experimental data obtained were analyzed in accordance with the method described in "C, Statistical Analysis" of the "General Experimental Method" above. B. Inhibition pattern of peptide for ACE: An appropriate amount of the peptide obtained in the above Example 2 was dissolved in deionized water to form a peptide solution having a concentration of 0.02 and 0.04 mM, respectively, and deionized water. It was used as a control group. Each concentration of the peptide solution was substantially determined by the method described in "Measurement of A, ACE Inhibitory Activity" of "General Experimental Method" above, except that the experiment was repeated 4 times, and each was The HHL concentrations used in the next measurement were 3.94, 1.97, 0.985, and 0.493 mM, respectively. The experimental results thus obtained were plotted as Lineweaver-Burk plots for analysis. A. Effect of concentration of peptide on ACE inhibitory activity: Figure 6 shows the ACE inhibitory activity of peptides of pepsin hydrolysate isolated from chlorella waste at various concentrations. As can be seen from Fig. 6, the ACE inhibitory activity increased as the peptide concentration increased, and the calculated IC5 〇 value was 29.6 μΜ. This result shows that pepsin 25 isolated from chlorella waste has a dose-dependent inhibition of ACE in the form of hydrolysate. B. Inhibition of peptides by ACE: Figure 7 shows a Lineweaver-Burk plot of peptides of pepsin hydrolysate isolated from chlorella waste at various concentrations for inhibition of ACE. As can be seen from Figure 7, the slope of the Lineweaver-Burk plot and the intercept intersecting the Y-axis increase with increasing peptide concentration, but the intercept intersecting the X-axis does not change. Therefore, the peptide of the pepsin hydrolysate separated from the chlorella waste belongs to the non-competitive inhibition of the inhibition mode of ACE, which means that regardless of the presence or absence of the ACE receptor, Peptides separated from pepsin hydrolysate of chlorella waste can be bound to ACE. Applicants hypothesize that peptides isolated from pepsin hydrolysate from chlorella waste may cause a conformational change in ACE to prevent ACE from converting the host into a product, so whether it is present at low or high concentrations The peptide can achieve the same ACE inhibition effect. Example 4. Effect of Absence, pH, and Gastrointestinal Protein ft|(gastrointestinal protease) on peptides isolated from chlorella waste of gastric protein plum hydrolysate for further investigation of temperature, pH, and gastrointestinal proteases The effect of the peptide of the pepsin hydrolyzate isolated from the chlorella waste of the present invention on the ACE inhibitory effect, the applicant used the peptide having the ACE inhibitory activity obtained in Example 2 to carry out the following experiment. Experimental method: A. Effect of temperature on peptide: 26 201043239 The peptide obtained in the above Example 2 was dissolved in deionized water to form a peptide solution having a concentration of 1%. The obtained peptide solution was divided into 4 groups and allowed to stand at 40, 60, 80, and 10 (TC for lapsed for several hours, and then according to the above "general experimental method," "A, ACE inhibitory activity measurement, The method was used to determine the ACE inhibitory activity of each group of peptide solutions.B. Effect of pH on the peptide: The peptide obtained in the above Example 2 was dissolved in deionized water to form a peptide solution having a concentration of 1%. The obtained peptide solution was divided into 5 groups of hydrazines and the pH was adjusted to 2, 4, 6, 8, and 10, respectively, and then allowed to stand at 37 t for 1 hour. Thereafter, according to the above "general experimental method," The method described in the measurement of ACE inhibitory activity to determine the ACE inhibitory activity of each group of peptide solutions. c. The effect of gastrointestinal phlegm on peptides: The effect of gastrointestinal proteases on peptides is mainly based on j Wu an(j X · Ding (2QQ2\Food Research International, 35:36Ί-375) The method described in the section was slightly modified and later evaluated. Briefly, 1 g of hydrazine was dissolved in 10 mL from the peptide obtained in Example 2 above. 0.1 M KC1-HC1 buffer solution (pH In 2.0), pepsin of o.oi g was then added to form a mixture. The resulting mixture was placed in a water bath having a temperature of 37 C for 3 hours. Thereafter, the mixture was The pH was adjusted to 7.8, and then heated to boiling for 1 俾 minutes to deactivate the pepsin. After the mixture was cooled, 〇.1 g of pancreatin was added and then placed in a The incubation was carried out in a water bath at a temperature of 37 ° C for 27 hours, 201043239 for 4 hours. After that, it was heated to boiling for 〇 minutes, and then centrifuged at 1 〇, _ g for 10 minutes. The obtained supernatant was obtained. The ACE inhibitory activity was determined according to the "General Test Method," "Measurement of A, ACE Inhibitory Activity," and the method described therein. Results: pH and temperature for pepsin hydrolysate isolated from chlorella waste The effect of the peptide was found from the experimental results: the peptide of the present invention was placed at a temperature of not _PH (PH2, 4, 6, 8 and 1 〇) and different temperatures (10) after that It has ACE inhibitory activity (data not shown). Applicants accordingly conclude that the peptide of the present invention has superior pH- and heat-stability (pH_ and "仏stability". In addition, related to gastrointestinal tract protein for the present invention The effect of the peptide was found by the experimental results: after the in vitro incubation with pepsin and trypsin, the peptide # ACE inhibitory activity of the present invention did not exhibit statistical significance compared with the control group (7)>;0.05) (data not shown). The applicant infers from this that the peptide of the present invention is resistant to gastrointestinal enzymatic enzymatic digestion, so that the peptide of the present invention can overcome the environmental pressure of the human digestive tract and exert its desired effect after ingestion. . From the above experimental results, it is known that the peptide of the pepsin hydrolyzate separated from the chlorella waste has excellent ACE inhibitory activity and good pH_and heat-stability, and thus is expected to be used for preparation for regulating blood pressure. Medicinals or dietary supplements. 28 201043239 Example 5. Peptide of pepsin hydrolysate isolated from chlorella waste The growth inhibitory effect of human gastric adenocarcinoma cell line AGS is evaluated in order to understand the separation according to the present invention. The pepsin of the chlorella waste. The peptide of the hydrolyzate has an effect on the proliferation and differentiation of the human gastric adenocarcinoma cell line AGS, and the applicant used the peptide having the ACE inhibitory activity obtained in Example 2 to carry out the following experiment. 〇 it test method · The following evaluation of the anti-incremental effect is carried out with reference to the method described in S. Katayama and Y. Mine (2007), J./G. First, human gastric adenocarcinoma AGS was inoculated into a 96-well plate (4×10 3 cells/well) containing RPMI medium (containing 10% fetal bovine serum), and cultured at 37 ° C for a duration of time. 24 hours. Thereafter, _ an appropriate amount of the peptide solution (the peptide obtained in the above Example 2 was dissolved in the deionized water) was added to a final concentration of 206, 244 and 306 μM, respectively, and then cultured for 72 hours. In addition, cells to which deionized water was added were used as a control group. Next, ΜΤΤ (20 μί / well) was added to each well and cultured for 4 hours. Thereafter, 200 μM of 100% dimethyl hydrazine (DMSO) was added to each well to dissolve ΜΤΤ-formazan. The absorbance of each well (〇D57Q) was read at a wavelength of 570 nm using a microplate reader. The cell growth inhibition rate was calculated by substituting the measured OD57 〇 value into the following formula: 29 201043239: A(%) = [(BC)/B]xl00 where: A = cell growth inhibition rate B = control The experimental data obtained by the OD57〇 value of the group OD OD57〇 value of the experimental group were analyzed according to the method described in “C, Statistical Analysis” of the above “General Experimental Method”. In addition, Applicants synthesized the same three experiments, namely PEP1 (SEQ ID NO: 2), PEP2 (SEQ ID NO: 3), and PEP3 (SEQ ID NO: 4), and the three peptides each had one. The amino acid sequence is identical to a portion of the amino acid sequence of the peptide of the present invention (SEQ ID NO: 1) (see Table 2). Table 2. Synthetic peptide peptide name amino acid sequence 之一1 Pro-Asn-Arg-Pro-Gln-Phe (SEQ ID NO: 2) with a portion of the amino acid sequence of the amino acid sequence of the peptide of the present invention. ΡΕΡ2 Val-Glu-Cys-Tyr-Gly-Pro (SEQ ID NO: 3) ΡΕΡ3 匚>^-丁71'-01丫-?1'〇-八811-八^(Sequence ID: 4) # Fig. 8 shows the cell growth inhibition rate of human gastric adenocarcinoma AGS after treatment with different concentrations of peptide. As can be seen from Fig. 8, the cell growth inhibition rate of AGS cells was positively correlated with the concentration of the peptide, and the calculated IC5Q value was 256.4 ± 1.2 μΜ. This result shows that the peptide of the present invention has a dose-dependent inhibitory effect on the growth of human gastric adenocarcinoma cell AGS. 30 201043239 In addition, the peptide of the present invention exhibited a superior growth inhibitory effect of human gastric adenocarcinoma AGS (data not shown) in comparison with the synthetic peptides PEP1, PEP2 and PEP3. This result shows that the sequence and structure of the peptide of the present invention may play an important role in the growth inhibitory effect against human gastric adenocarcinoma AGS. Example 6. Evaluation of inhibitory effect of peptides of gastric peptone hydrolysate isolated from chlorella waste for inducible NO production 〇 Experimental method · The following inhibitory effect on inducible NO production is a reference X. Yang W a/. (2008), Food CAe/w., 106: 269-276 The method described in the paper was evaluated with minor modifications. First, mouse macrophage RAW 264.7 was inoculated into a 96-well plate (5 x 10 4 cells/well) containing RPMI medium (containing 10% fetal calf serum), and cultured at 37 ° C for 4 hours. Thereafter, 20 pL of LPS (50 pg/mL) was added and an appropriate amount of peptide solution (the peptide obtained in the above Example 2 was dissolved in deionized water) was added to a final concentration of 9.9, 19.9 and 99.3 μM, respectively. . In addition, cells to which no peptide solution was added were used as a control group. Next, after incubation at 37 ° C for 24 hours, 80 μί of the supernatant was mixed with 160 μg of Griess reagent [1% of sulfanilamide, 0.1% at room temperature. N-naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid were mixed and reacted for 20 minutes. The absorbance (ODho) of 31 201043239 at a wavelength of 540 nm was measured by an ELISA reader (p〇werwave xs, Biotek, Vermont, USA), and the obtained 〇d54() value and sodium nitrite (NaN〇2) were obtained. The quantitative standard curve is aligned to calculate the concentration of the nitrite product. The NO production inhibition rate was calculated by substituting the measured nitrite product concentration into the following formula: A (%) = [(BC) / B] xl00 where: A = NO production inhibition rate B = control The concentration of the nitrite product of the group C = the concentration of the nitrite product of the experimental group. Figure 9 shows the inhibition rate of NO formation measured by the treatment of different concentrations of peptides of the small gas giant cell raw 264.7. As can be seen from Fig. 9, the inhibition rate of NO production of mouse macrophage RAW 264.7 increased with increasing peptide concentration, and the calculated ICS0 value was 42·4 μΜ. This result shows that the peptide of the pepsin hydrolyzate separated from the chlorella waste has a dose-dependent inhibitory effect on induced sputum production. From the results of the experiment, it is understood that the pepsin hydrolysate isolated from the chlorella waste of the present invention has an effect of inhibiting the formation of sputum. Because the production of sputum is associated with the development of inflammatory symptoms. Therefore, the present invention is also expected to have an anti-inflammatory effect and can be used for the preparation of a pharmaceutical or dietary supplement for treating or preventing an inflammatory disease. Example 7. Isolation of peptides with antioxidant activity Identification of the pepsin hydrolysate of chlorella waste has been found to have antioxidant activity in a prior study by the applicant. Because of this, in this example, the applicant has a form of antioxidant 32 201043239 which is active in the gastric protein hydrolysate of chlorella waste. Experimental method: A. Preparation of gastric peptone hydrolysate of chlorella waste: 10 g of chlorella waste was mixed in 100 mL of deionized water to form a concentration of 10% (w/v). Chlorella waste solution. Dissolve

液的pH值調整至pH 2.0 ’繼而加入適量的胃蛋白酶至一最 終濃度為0.2% (w/v)，藉此而形成一反應混合物。所形成的 反應混合物被置於50。(：下進行水解歷時15小時。之後，將 =溶液的PH值調整至PH 7.0,藉此將胃蛋白酶去活化。接 著，在10,000 rpm下予以離心歷時2〇分鐘，之後，收取上 清液而得到小球藻廢棄物的胃蛋㈣水解物。 所得到的胃蛋白酶水解物是藉由使用20% SDS-聚丙烯 ^ ^ ^ # t ^ (sodium dodecyl sulfate-polyacrylamide gel _Γ〇Ρίΐ〇Γ_，SDS_PAGE)來進行分析，並且依照上面“一 般實驗方法”的“B、Tr〇inY米具 > 贫 §量抗虱化物能力分析，，當中所 述方法來測定抗氧化活性。 斤仔、，口果刀別被顯示於圖10以 得到的胃蛋⑽水解對小球藻廢棄物進行水解所 ，小抹、落故直 AGE電泳圖。從圖10可見 解的小«廢棄物所具者是較小解的物的刀子量相較於未經水 圖11顯示藉由使田 解所得到的胃蛋白嗨水解：’對小球藻廢棄物進行水 小球藻廢棄物的胃蛋白 ' 几氧化活性。從圖11可見， 水解物顯示出具有高的對抗ams 33 201043239 自由基的抗氧化活性，而未經水解的小球藻廢棄物則沒有 可觀察到的抗氧化活性。因& ’在下面的實驗中，申請人 將從小«廢棄物的胃蛋㈣水解物中進—步分離出 抗氧化活性的肽。 八 B、硫酸銨分級分離處理： 將適量的硫酸錄緩慢加人至上面A項當中所得到的小 球藻廢棄物的胃蛋白酶水解物中，直到所形成的溶液具有 一為4G%的硫酸㈣和度。接著，在1〇，_ g下離心歷時 〇刀鐘而得到-上清液以及—沉搬物。該沉澱物被散浮於 1〇1^的去離子水中’藉此而得到對應於G〜4G%的硫酸錢飽 和度的分離部分F1。 另外所得到的上清液被添加適量的碗酸録，直到所 形成的溶液具有一為80%的硫酸銨飽和度。之後，在 10,000 g下離心歷時20分鐘’繼而移除上清液。所得到的 沉搬物被散浮於U) mL的去離子水中，藉此而得到對應於 40〜80%的硫酸銨飽和度的分離部分F2。 接著，分離部分F1與F2分別被倒入一具有一為1〇⑽ 道耳頓的分子量截斷值的透析膜（CelluSep)中並置於去離子 水中進行透析。經透析的分離部分是依照上面“一般實驗方 法”的“B、Trolox當量抗氧化物能力分析，，當中所述方法來 測定抗氧化活性。 實驗結果發現.分離部分F2 (對應於4〇〜8〇%的硫酸銨 飽和度）所具有的抗氧化活性相較於分離部分F丨（對應於 〇〜40%的硫酸銨飽和度）所具者是較高的（約為24倍）。因此 34 201043239 ，將對應於40〜80%的硫酸銨飽和度的分離部分F2冷凍乾 燥以供進行下面的分析。 C、 凝膠過濾層析：The pH of the solution was adjusted to pH 2.0' and then an appropriate amount of pepsin was added to a final concentration of 0.2% (w/v), thereby forming a reaction mixture. The resulting reaction mixture was placed at 50. (The hydrolysis was carried out for 15 hours. Thereafter, the pH of the solution was adjusted to pH 7.0, whereby pepsin was deactivated. Then, centrifugation was carried out at 10,000 rpm for 2 minutes, after which the supernatant was collected. The stomach egg (tetra) hydrolyzate of the chlorella waste is obtained. The obtained pepsin hydrolyzate is obtained by using 20% SDS-polypropylene ^ ^ ^ # t ^ (sodium dodecyl sulfate-polyacrylamide gel _Γ〇Ρίΐ〇Γ_, SDS_PAGE ) to carry out the analysis, and according to the "general experimental method" above, "B, Tr〇inY rice utensils> poor § anti- mash resistance analysis, the method described therein to determine the antioxidant activity. The knife is shown in Figure 10 to obtain the hydrolysis of the chlorella waste by the hydrolysis of the stomach egg (10), and the AGE electrophoresis pattern of the small smear and the falling AGE. It can be seen from Fig. 10 that the small waste is smaller. The amount of the knives of the solution was compared with that of the water peptone obtained by degrading the soil without the water diagram: 'the gastric protein' oxidative activity of the chlorella waste of the chlorella waste. As can be seen from Figure 11, the hydrolyzate is shown to have The anti-oxidant activity of the free radicals against ams 33 201043239, while the unhydrolyzed chlorella waste has no observable antioxidant activity. Because & 'In the following experiment, the applicant will be from small «waste The stomach egg (4) hydrolysate further separates the peptide with antioxidant activity. Eight B, ammonium sulfate fractionation treatment: The appropriate amount of sulfuric acid is slowly added to the pepsin obtained from the chlorella waste obtained in item A above. In the hydrolyzate, until the solution formed has a sulfuric acid (four) and degree of 4 G%. Then, the crucible clock is centrifuged at 1 Torr, _g to obtain a supernatant and a sinking object. The precipitate is Dissolved in 1 〇 1 ^ deionized water 'by this to obtain a separation portion F1 corresponding to G 4 g% sulfuric acid saturation. The obtained supernatant was added with an appropriate amount of bowl acid until it was formed. The solution has an 80% ammonium sulfate saturation. Thereafter, it is centrifuged at 10,000 g for 20 minutes. Then the supernatant is removed. The resulting sink is dispersed in U) mL of deionized water. This results in a saturation of 40 to 80% ammonium sulfate. The separated portion F2. Next, the separated portions F1 and F2 are respectively poured into a dialysis membrane (CelluSep) having a molecular weight cutoff value of 1 〇(10) Dalton and placed in deionized water for dialysis. The antioxidant activity was determined according to the "B, Trolox equivalent antioxidant capacity analysis" of the "general experimental method" above. As a result of the experiment, it was found that the separation portion F2 (corresponding to 4 〇 to 8 〇% of ammonium sulfate saturation) has an antioxidant activity as compared with the separation portion F 对应 (corresponding to 〇 40% of ammonium sulfate saturation). The one is higher (about 24 times). Therefore, 34 201043239, the separated portion F2 corresponding to 40 to 80% of ammonium sulfate saturation was freeze-dried for the following analysis. C, gel filtration chromatography:

將上面B項當中所得到的經冷凍乾燥之分離部分F2溶 解在5 mL的去離子水中，並依據下面的操作條件來進行凝 . 膠過濾層析：分析管柱為一經20 mM Tris-HCl緩衝溶液（pH 7_8)預平衡的 Sephacryl S-100 high HR 管柱（長度：2_6 cm X 70 cm);洗提液為20 mM Tris-HCl緩衝溶液（pH 7.8);流速 〇 被控制為1.5 mL/min ;偵測波長被設定為220 nm。於洗提 時，將每6 mL的洗出物收集於一個試管内。 依據所得到的洗提圖形來收集各波峰所對應的洗出物 ，繼而依照上面“一般實驗方法”的“B、Trolox當量抗氧化 物能力分析”當中所述方法來測定抗氧化活性。 實驗結果發現：在凝膠過濾層析的洗提圖形中有2個 波峰被觀察到。分別收集該等波峰所對應的洗出物而得到 分離部分G1與G2 (參見圖12)。測定分離部分G1與G2的 〇 抗氧化活性，結果發現：分子量較低的分離部份G2所具有 的抗氧化活性相較於分離部份G1所具者是較高的（約為2.5 倍）（數據未顯示）。因此，將分離部分G2冷凍乾燥以供進行 下面的分析。 D、 陰離子交換層析： 將上面C項當中所得到的經冷凍乾燥之分離部分G2溶 於5 mL的去離子水中，並參照上面實施例2的“C、陰離子 交換層析”當中所述方法來進行陰離子交換層析，不同之處 35 201043239 在於：偵測波長被設定為220 nm。 依據所得到的洗提圖形來收集各波峰所對應的洗出物 ，繼而依照上面“一般實驗方法”的“B、Trolox當量抗氧化 物能力分析”當中所述方法來測定抗氧化活性。 實驗結果發現：在洗提液的NaCl濃度為0 Μ時所收集 到的分離部分具有較高的抗氧化活性（數據未顯示）。因此， 收集該具有較高的抗氧化活性的分離部分，繼而予以冷凍 乾燥以供進行下面的分析。 Ε、逆相高效能液相層析： 將上面D項當中所得到的經冷凍乾燥之分離部分溶於1 mL的去離子水中，並依據下面的操作條件來進行逆相高效 能液相層析：儀器型號為 Agilent 1100 (Agilent Technologies Inc. CA, USA);分析管柱為 Inertsil ODS-3 C18半製備級管柱（10 mm x 250 mm);洗提液為具有一乙 腈梯度（acetonitrile gradient)(l5 至 35%，30 分鐘）的含有 0.1 % TFA之水溶液；流速被控制為2 mL/min ;偵測波長被 設定為220 nm。 依據所得到的洗提圖形來收集各波峰所對應的洗出物 ，繼而依照上面“一般實驗方法”的“B、Trolox當量抗氧化 物能力分析”當中所述方法來測定抗氧化活性。 實驗結果發現：在滯留時間約為23分鐘時偵測到一波 峰（參見圖13)，並且該波峰所對應的洗出物（亦即分離部分 P2)具有抗氧化活性。將分離部分P2冷凍乾燥而得到具有抗 氧化活性的肽。 36 201043239 所得到的具有抗氧化活性的肽是藉由使用一 Agilent 6510 Q-TOF 質譜儀（Agilent 6510 Q-TOF mass spectrometer)(Agilent Technologies Inc. CA，USA)來確認純 度。結果顯示：在本實驗中所得到的肽的純度大於98%。 因此，將所得到的肽進行下面的胺基酸序列分析。 F、胺基酸序列分析： 在上面E項當中所得到的肽是藉由使用 Applied Biosystems Procise 494蛋白質定序儀並參照廠商所提供的 〇 操作指引來進行胺基酸序列分析。分析結果顯示：在本實 施例中所得到的具有抗氧化活性的肽的胺基酸序列與上面 實施例2中所得到的具有ACE抑制活性的肽所具者相同。 另外’申請人為了驗證在本實施例中所得到的肽確實 具有抗氧化活性，一具有如序列辨識編號：1所示的胺基酸 序列的肽被合成出’並且依照上面“ 一般實驗方法，，的“B、 Trolox當量抗氧化物能力分析，，當中所述方法來測定抗氧化 活性。結果顯示：合成的肽亦具有如同本實施例中所得到 Ο 的肽的抗氧化活性（數據未顯示）。 實施例8.分離自小球藻廢棄物的買蛋白晦水解物的肽的抗 氧化效用評估 為瞭解依據本發明之分離自小球藻廢棄物的胃蛋白酶 水解物的肽對於不同來源的自由基[包括：ABTS自由基、 DPPH自由基、過氧自由基（per〇xyi radicais)、經基自由基 (hydroxyl radicals)以及超氧自由基（super〇xide radicals)]的 抗氧化效用’申請人使用實施例7當中所得到的肽來進行 37 201043239 下面的實驗。 實驗方法'· A、Trolox當量抗氣化物能力分析： 1. 抗氧化物溶液（antioxidant solutions)以及ABTS溶液的製 備： 在本實驗中，上面實施例7中所得到的肽、Trolox以 及抗壞血酸分別被配製於去離子水中，而BHT被配製於1% DMSO中以獲得具有不同濃度的抗氧化物溶液。 另外，ABTS 溶液是參照 M.J.T.J. Arts d a/. (2004)(同 上述）當中所述方法來製備。 2. 實驗程序： 各個抗氧化物溶液的TEAC分析是參照上面“一般實驗 方法”的“B、Trolox當量抗氧化物能力分析”當中所述方法 來進行，並且在混合物中之抗氧化物濃度被顯示在下面的 表3中。 表3.抗氧化物的濃度 抗氧化物 濃度（μΜ) 肽 3.56 ' 7.12 ' 14.24 ' 28.48 BHT 3.56、7.12、14.24、28.48 Trolox 11.90 ' 23.81 ' 35.71 抗壞血酸 7.44 ' 14.87 ' 29.75 此外，這4種抗氧化物的清除50%的ABTS自由基的 濃度（IC5〇)被計算出。所得到的實驗數據是依照上面“一般實 驗方法”的“C、統計學分析”當中所述方法來進行分析。 38 201043239 B ' DPPH 自由基清除活性分析（DPPH radicals scavenging activity assay): 1. 抗氧化物溶液以及DPPH溶液的製備： 在本實驗中，上面實施例7中所得到的肽以及抗壞血 酸分別被配製於去離子水中，而BHT以及Trolox分別被配 . 製於1% DMSO以及無水酒精中以獲得具有不同濃度的抗氧 化物溶液。 另外，DPPH 溶液是參照 S.J. Huang and J.L. Mau 〇 (2006)，5W. Tec/mo/., 39:707-716 當中所述方法來製備 ο 2. 實驗程序： 各個抗氧化物溶液的DPPH自由基清除活性分析是參考 S.J. Huang and J_L. Mau (2006)(同上述）當中所述方法來進行 。簡言之，將不同濃度的抗氧化物溶液（4 mL)分別與DPPH 溶液（1 mL)混合以形成含有不同濃度之抗氧化物（參見表4) * 的混合物。之後，將所形成的混合物靜置於黑暗中進行反 〇 應歷時40分鐘，繼而在一為517 nm的波長下測定吸光值 (OD517) 〇另外，對照組是以製備抗氧化物溶液時所使用的 溶劑來取代抗氧化物溶液。 表4.抗氧化物的濃度 抗氧化物 濃度（μΜ) 肽 29.9083 、 59.8167 、 119.6 BHT 454.55、909.09、1818.18 Trolox 11.99、23.97、31.96、95.88 抗壞血酸 10.68、21.35、42.70、85.41 39 201043239 各個抗氧化物溶液的DPPH自由基清除活性是藉由將所 測得的OD517數值代入下列公式而被計算出： A(%)= [(B-C)/B]xl00 其中：A=DPPH自由基清除活性 B=對照組的OD517數值 〇抗氧化物溶液的OD517數值 此外，這4種抗氧化物的清除50%的DPPH自由基的 濃度（IC5G)被計算出。所得到的實驗數據是依照上面“一般實 驗方法”的“C、統計學分析”當中所述方法來進行分析。 C、經基自由基清除能力分析（Hydroxyl radicals scavenging ability assay): 1. 抗氧化物溶液以及反應物溶液的製備： 在本實驗中，上面實施例7中所得到的肽以及Trolox 分別被配製於去離子水中，而BHT被配製於1% DMSO中 以獲得具有不同濃度的抗氧化物溶液。 另外，反應物溶液（含有1.0 mL的3 μΜ IBG、0.1 mL 的 1.0 mM FeS04、1.6 mL 的 3% H2〇2 以及 0.05 mL 的 10 mM EDTA)是參照 C.H. Tsai <2厂（2001), /· JgTic· Food C/iem·, 49:2137-2141當中所述方法來製備。 2. 實驗程序： 各個抗氧化物溶液的經基自由基清除能力分析是參考 C.H. Tsai ei a/. (2001)(同上述）當中所述方法來進行。簡言 之，將不同濃度的抗氧化物溶液（10 μι)分別添加至上述反 應物溶液中以形成含有不同濃度之抗氧化物（參見表5)的混 40 201043239 合物’繼而使用一配備有一高感度 >(貞測器的BJL-ultraweak 化學螢光分析儀（BJL-ultraweak chemiluninescence analyser, Jye Horn Co. Taiwan)來偵測化學榮光（chemiluninescence, CL)的變化。另外，對照組是以製備抗氧化物溶液時所使用 的溶劑來取代抗氧化物溶液。 表5.抗氧化物的濃度 抗氧化物 濃度（μΜ) 肽 1.99、3.99、5.97、7.96、9.95 ΒΗΤ 16.53、33.06、49.59、66.12 Trolox 1.45、2.91、4.36、5.81 各個抗氧化物溶液的經基自由基清除活性是藉由將所 測得的化學螢光數（CL counts)代入下列公式而被計算出： A(%)= [(B-C)/B]xl00 其中：A=羥基自由基清除活性 B=對照組的化學螢光數 〇抗氧化物溶液的化學螢光數 此外，這3種抗氧化物的清除50%的經基自由基的濃 度（IC5〇)被計算出。所得到的實驗數據是依照上面“一般實驗 方法”的“C、統計學分析”當中所述方法來進行分析。 D、超氧自由基清除能力分析（Superoxide radicals scavenging ability assay) ' 1.抗氧化物溶液以及反應物溶液的製備： 在本實驗中，上面實施例7中所得到的肽以及Trolox 分別被配製於去離子水中，而BHT被配製於1% DMSO中 41 201043239 以獲得具有不同濃度的抗氧化物溶液。 另外’反應物溶液[含有1.0 mL的2.〇 mM光澤精 (lucigenin)、0.〇5 mL 的 1.0 Μ 精胺酸、〇 〇5 机的 i 4 _ 丙酮醛以及1.0 mL的PBS緩衝液（ΡΗ 7·4)]是參照c H Tsai ei fl/. (2003)，/·处Food CAem.，51:58-62 當中所述方法 來製備。 2.實驗程序： 各個抗氧化物溶液的超氧自由基清除活性分析是參考 C.H_ Tsai ei a/· (2003)(同上述）當中所述方法來進行。簡言 之’將不同濃度的抗氧化物溶液（10 分別添加至上述反 應物溶液中以形成含有不同濃度之抗氧化物（參見表6)的混 合物，繼而使用一配備有一高感度偵測器的BJL-Ultraweak 化學螢光分析儀來偵測化學螢光的變化。另外，對照組是 以製備抗氧化物溶液時所使用的溶劑來取代抗氧化物溶液 表6 ·抗氧化物的濃度 抗氧化物 濃度（μΜ) 肽 3.38、6.75、10.13、13.50、16.88 BHT 44.35、88.69、133.04、177.38、221.73 Trolox 19.49、38.98、58.47 各個抗氧化物溶液的超氧自由基清除活性是藉由將所 測得的化學螢光數代入下列公式而被計算出： A(%)= [(B-C)/B]xl〇〇 其中：A=超氧自由基清除活性 42 201043239 B=對照組的化學螢光數 C=抗氧化物溶液的化學螢光數 另外，這3種抗氧化物的清除50%的超氧自由基的濃 度（IC5〇)被計算出。所得到的實驗數據是依照上面“一般實驗 方法，，的“C、統計學分析，，當中所述方法來進行分析。 E、氧自由基吸收能力分析[〇xygen radical absorbance capacity (ORAC) Assay]： 1.抗氧化物溶液、螢光素溶液以及aaph溶液的製備： 〇 在本實驗中，上面實施例7中所得到的肽、BHT、抗壞 血酸以及Trolox分別被配製於75 mM的磷酸緩衝鹽水溶液 (phosphate buffered saline, PBS)中以得到抗氧化物溶液。 Trolox被使用作為標準品。 另外，96 nM的螢光素溶液以及320 mM的AAPH溶液 是藉由將適量的螢光素以及AAPH分別配於75 mM的PBS 中而被獲得。 ' 2.實驗程序： 〇 各個抗氧化物溶液的ORAC分析是參考D. Alberto w α/. (2004)，J. JgWc. Food CAem·, 52:48-54 當中所述的方法 並略作修改後來進行。簡言之，將20 pL的抗氧化物溶液以 及150 pL的螢光素溶液加入至微量滴定盤（microplate)的井 内，繼而於37°C下進行預培育歷時5分鐘。之後，加入30 μί的AAPH溶液以起始反應，此時所形成的混合物中的抗 氧化物濃度被顯示於下面的表7中。 43 201043239 表7.抗氧化物的濃度 抗氧化物 濃度（μΜ) 肽 3.7 ' 7.5 BHT 29.9 Trolox 10 抗壞血酸 31.2 接著，立即將微型盤置於讀取儀（reader)中並以一為2 分鐘的時間間隔來紀錄螢光值，總共歷時130分鐘，藉此 而得到各個抗氧化物溶液的螢光衰退曲線（fluorescene decay curves)。另外，對照組是以75 mM的PBS來取代抗氧化物 溶液。 .结耒·· A、Trolox當量抗氧化物能力分析： 圖14顯示本發明的肽、BHT、Trolox以及抗壞血酸的 ABTS自由基清除活性。從圖14可見，這4種抗氧化物對 於ABTS自由基的清除活性皆具有劑量-依賴性。此外，就 清除ABTS自由基的能力來看，最高者為本發明的肽（IC50 數值為9·8±0·5 μΜ)，其次為抗壞血酸（IC5〇數值為30.0±0·8 μΜ)以及Trolox (IC5〇數值為32·5±1·3 μΜ)’而合成的抗氧 化物BHT是最低的（IC5〇數值為57.4±2·3 μΜ)。 Β、DPPH自由基清除活性分析： 圖15顯示本發明的肽、Trolox以及抗壞血酸的DPPH 自由基清除活性。從圖15可見，本發明的肽在濃度為 29.9083以及59.8167 μΜ的情況下對於DPPH自由基的清除 44 201043239 活性皆具有劑量-依賴性。此外，就清除DPPH自由基的能 力來看，最高者為Trolox (IC5〇數值為23.0±1.8 μΜ)，其次 為抗壞血酸（IC5〇數值為44.0 μΜ)以及本發明的肽（IC5〇數值 為58.0±1·2 μΜ)，而合成的抗氧化物BHT是最低的（IC50數 值為968·3±2.6 μΜ)(圖式未顯示）。 另外，類似的先前研究顯示：源自牛蛙皮膚（bullfrog skin)的肽具有一 IC5〇 數值為 16.1 μΜ (Z.J. Qian α/. (2008a), e/oresowr. JlecAwo/., 99:1690-1698) ’ 而源自酷·蛋白 (casein)的肽具有一 IC50 數值為 98 μΜ (K. Suetsuna α/. (2000), /· iVwiW. 5/ocAem.，11:128-131)。將本發明的肽與該 等習知的肽作一比較後發現，本發明的肽具有中等的對抗 DPPH自由基的能力。 C、 幾基自由基清除能力分析： 圖16A顯示本發明的肽、BHT以及Trolox的羥基自由 基清除活性。從圖16A可見，這3種抗氧化物對於羥基自 由基的清除活性皆具有劑量-依賴性。此外，就清除經基自 由基的能力來看，最高者為Trolox (IC5〇數值為2.0±0.3 μΜ) ，其次為本發明的肽（IC5〇數值為8.3±0.15 μΜ)，而BHT (IC5〇數值為44.4±1.6 μΜ)是最低的。 D、 超氣自由基清除能力分析： 圖16Β顯示本發明的肽、ΒΗΤ以及Trolox的超氧自由 基清除活性。從圖16B可見，本發明的肽的超氧自由基清 除活性會隨著濃度的增加而急劇的升高。此外，就清除超 氧自由基的能力來看，最高者為本發明的肽（IC5〇數值為7.5 45 201043239 ±0.12 μΜ)，其次為 Trolox (IC5〇 數值為 24·8±0·4 μΜ)，而 ΒΗΤ (IC5〇數值為218_1±2.1 μΜ)是最低的。 另外，類似的先前研究顯示：源自酷蛋白的肽具有一 IC50 數值為 79.2 μΜ (K. Suetsuna et al. (2000)，同上述）、 源自牡蠣蛋白水解物的肽具有一1C5Q數值為78.971111^(2：.】· Qian a/. (2008b)，价0asowr· rec/iwo/.，99:3365-3371)，以 及2種源自於大烏賊（giant squid)的肌肉蛋白水解物的肽分 別具有 IC5〇 數值為 669.34 以及 573.83 μΜ (N. Rajapakse ei a/· (2005)，同上述）。將本發明的肽與該等習知的肽作一比 較後發現，本發明的肽具有最低的1C5()數值（亦即在低濃度 下就具有良好的清除超氧自由基的能力）。 E、氧自由基吸收能力分析： 圖17顯示本發明的肽、Tr〇l〇x、BHT以及抗壞金酸在 ORAC分析中所得到的螢光衰退曲線。從圖17可見’本發 明的肽在螢光衰退的抑制上具有一劑量-依賴的升高。此外 ，就對抗過氧自由基（peroxy1 radicals)的能力來看，最高者 為本發明的肽，其次為Trolox，再其次為抗壞血酸，而 BHT是最低的。過氧自由基在脂質過氧化反應（lipid peroxidation)中會被形成’因此’申請人推論：本發明的肽 可藉由清除過氧自由基來終止脂質過氧化反應的自由基鍵 反應（free radical chain reaction)。 實施例9·具有抗氧化活性的肽對於羥基自由基-誘發的質《 DNA 傷害（hydroxyl radicals-induced plasmid DNA damage)的保護效用的評估 46 201043239 為瞭解依據本發明之分離自小球藻廢棄物的胃蛋白酶 水解物的肽對於由羥基自由基所誘發的DNA傷害的保護效 用，申請人使用實施例7當中所得到的肽來進行下面的實驗 〇 實嫌方法· • 本實驗是參考Z.J. Qian ei a/· (2008b)(同上述）當中所述 的方法並略作修改後來進行。簡言之，將適量的從上面實 施例7中所得到的肽溶於1 mL的去離子水中以形成濃度分 〇 別為10.6、42.4以及84.9 μΜ的肽溶液。另外，對照組是 以去離子水來取代肽溶液。 將4 μί的肽溶液與8 pL的質體pET-28a、3 pL的2 mM FeS〇4以及4pL的0.06 mM H2O2充分混合以形成一反 應混合物，繼而在37°C下進行預培育歷時30分鐘。之後， 以1%壤脂糖凝膠電泳（agarose gel electrophoresis)來進行分 析，繼而以SYBR Green來染色，然後於紫外光下作觀察並 照相。正常對照組（normal control)是不作任何處理的質體 O pET-28a，而正對照組(positive control)是經 FeS〇4 與 Η2〇2 處理的質體pET-28a。 結果： 圖18是一電泳膠片圖，其顯示經羥基自由基-誘發氧化 性傷害（hydroxyl radicals-induced oxidative damage)的質體 DNA以不同濃度的肽予以處理後的瓊脂糖凝膠電泳分析結 果，其中〇C DNA表示：呈現開環（open circular，OC)構形 的DNA ; SC DNA表示：呈現超螺旋（super coil，SC)構形的 47 201043239 DNA，徑1疋不作任何處理的質體pET 28a (正常對照組)； 仫2疋使用FeS04與h2〇2來誘發氧化性傷害的質體ρΕτ· 28a (正對照組）；以及徑3隸5是分別以濃度為84 9、 42.4以及10.6 μΜ㈣來處理經祕自由基誘發氧化性傷 害的質體PET_28a。實驗結果顯示，隸基自由基來誘發氧 化性傷害會使得質體DNA由超螺旋構形轉變成開環構形（參 見徑2)，但是，本發明的肽可有效地防止因為氧化性傷害 所造成的DNA構形的變化並且保護效用會隨著濃度的增加 而升高（參見徑3至徑5)。 實施例1G.具有抗氡化活性的肽的細跑毒性分析以及對於 帶有H2〇2·誘發的細胞傷害（H2〇2_induced cell damage)的人類胃腺癌細胞agS的保護效用的 評估 為瞭解依據本發明之分離自小球藻廢棄物的胃蛋白酶 水解物的肽對於由4〇2所誘發的細胞傷害的保護效用以及 對於細胞的毒性作用，申請人使用實施例7當中所得到的 肽來進行下面的實驗。 實驗方法： A本發明的肽對於1|2〇2·誘發的細胞傷害的保護效用的評 估： 將AGS細胞接種至含有RPMI培養基（含有1〇%胎牛血 ’月）的96-井培養盤（4χ1〇3細胞/井）内，繼而在37〇c下進行培 養歷時24小時。接著，加入適量的肽溶液（將上面實施例7 中所得到的肽配於去離子水中）至一最終濃度分別為〇 〇2、 48 201043239 0.04、0.08以及0.15 mM，繼而進行培養歷時2小時。之後 ，加入20 pL的H2〇2 (最終濃度為0.03 mM)並進行培養歷 時2小時。在經H2〇2處理的第2小時，以位相差顯微鏡 (phase-contrast micro scope) (Nikon)來觀察培養物的細胞型態 。正常對照組（normal control)是不作任何處理的AGS細胞 . ，而負對照組（negative control)是經0.03 mM H2〇2處理的 AGS細胞。 接著，將MTT (20 μί/井）加入至各井内並進行培養歷 〇 時4小時。之後，將200 μΐ之100% DMSO添加至各井中 ，俾以溶解出ΜΤΤ-甲臢。於570 nm的波長下以一微量滴 定盤讀取儀來讀取各井的吸光值（〇D57G)。細胞存活率是藉 由將所測得的〇D57〇數值代入下面的公式而被計算出： A(%) = (C/B)xl00 其中：A=細胞存活率 B=正常對照組的OD57〇數值 ' C=實驗組或負對照組的OD570數值 〇 所得到的實驗數據是依照上面“一般實驗方法”的“C、 統計學分析”當中所述方法來進行分析。 B、本發明的肽對於人類正常肺細胞WI-38的細胞毒性測試 將WI-38細胞接種至含有DMEM培養基（含有10%胎牛 血清）的96-井培養盤（3xl03細胞/井）内，繼而在37°C下進行 培養歷時24小時。接著，在各井中加入適量的肽溶液（將上 面實施例7中所得到的肽配於去離子水中）至一最終濃度分 49 201043239 別為0.02、〇_〇4、0.08以及〇_15 mM，並予以培養歷時72 小時。未經肽處理的細胞被使用作為對照組。 接著，將MTT (20 gL/井）加入至各井内並進行培養歷 時4小時。之後’將200 μΐ之100% DMSO添加至各井中 ，俾以溶解出ΜΤΤ-甲臢。於570 nm的波長下以一微量滴 定盤讀取儀來讀取各井的吸光值（〇D57〇)。細胞存活率是藉 由將所測得的〇D57〇數值代入下面的公式而被計算出： A(%) = (C/B)xl00 其中：A=細胞存活率 對照組的OD57〇數值 C=實驗組的〇D57〇數值 所得到的實驗數據是依照上面“一般實驗方法，，的“C、 統計學分析”當中所述方法來進行分析。 潜求.· 圖19A至圖19C分別顯示未經任何處理、經h2〇2處理 以及經H2〇2與本發明的肽處理的ags細胞的型態。從圖 19A至圖19C可見，未經處理的AGS細胞具有完整的細胞 型態，而經H2〇2處理的細胞顯示出明顯的氣泡（bubbles)。 然而，AGS細胞在被處理以H2〇2之前，若預先與本發明的 肽進行培育歷時2小時，由Η"2所誘發的細胞傷害會被明 顯地降低。這個結果顯示：本發明的肽對於h2〇2誘發的細 胞傷害的具有保護效用。 圖20顯示人類胃腺癌細胞AGS經不同濃度的肽處理之 後的細胞存活率。從圖20可見，帶有ha誘發細胞傷害 50 201043239 的AGS細胞的存活率僅約20%，然而，預先與本發明的肽 進行培育的AGS細胞在經過H202-誘發細胞傷害之後仍具 有優越的存活率，並且它們的存活率會隨著肽濃度的增加 而提高。這個結果顯示：分離自小球藻廢棄物的胃蛋白酶 水解物的肽對於由H202所誘發的細胞傷害具有一劑量-依賴 . 性的保護效用。 . 另外，申請人亦探討本發明的肽對於人類正常肺細胞 WI-38的細胞毒性，實驗結果顯示：wi-38細胞以不同濃度 〇 的肽予以處理後沒有生長抑制的現象被觀察到，因此本發 明的狀對於人類正常肺細胞WI-3 8不具有細胞毒性效用 (cytotoxicity effect)(數據未顯示）。 從上述的實驗結果可知，本發明的分離自小球藻廢棄 物的胃蛋白酶水解物的肽具有優異的抗氧化效用。因此， 本發明的肽被預期可供用於製備一用來預防由氧化性傷害 所造成的疾病[諸如動脈粥樣硬化（atheroscierosis)、冠狀動 脈心臟病（coronary heart disease)、癌症、阿茲海默症、老 〇 化、胃潰瘍等]的醫藥品或膳食補充品。 於本案說明書中被引述之所有文獻資料以及專利文件 以它們的整體被併入本案作為參考資料。若有所衝突時， 本案的詳細說明（包含界定在内）將佔上風。 雖然本發明已參考上述特定的具體例被描述，顯著地 在不背離本發明之範圍和精神之下可作出很多的修改和變 化。因此意欲的是，本發明僅受如隨文檢附之申請專利範 圍所示者之限制。 51 201043239 【圖式簡單說明】 圖1A與圖1B分別顯示小球藻廢棄物經不同的蛋 水解之後所測得的蛋白質產率以及ACE抑制活 白嗨 ，1王，其中“少 ”表示 ρ<0·05 ; 圖2顯示小球藻廢棄物的胃蛋白梅水解物經硫酸錢八 級分離處理後所得到的分離部分的ACE抑制活性其 、 至A4分別表示對應於〇〜2〇%、2〇〜4〇%、4〇〜的％^A1 60〜80%的硫酸錄飽和度的分離部分； 及 圖3顯示在本案實施例2的凝膠過濾層析中所得到 分離部分B1與B2的ACE抑制活性； 的 圖4A顯示在本案實施例2當中所進行的陰離子交 析的洗提圖形’其中C1與C2分別表示所得到的分離部分 :箭頭是示意分離部分的範圍；以及虛線表示Ν&α梯度. 圖4B顯示在本案實施例2的陰離子交換層析中所^ 的分離部分C1與C2的ACE抑制活性； 圖5A及圖5B分別顯示在本案實施例2當中使用 mensii ODS-3 C18半製備級管柱以及使用邮灿卿仙 C8管柱來進行逆相高效能液相層析的洗提圖形，其中⑴ 與Η分別表示所得到的分離部分；以及箭頭是示意分離部 分的範圍； 圖6顯示分離自小球藻廢棄物的胃蛋白崎水解物的肽 在不同濃度下的ACE抑制活性； 圖』示刀離自小球藻廢棄物的胃蛋白峰水解物的狀 在不同濃度下對於ACE的抑制活性的心爾心歸圖 52 201043239 圖8顯示人類胃腺癌細胞AGS經不同濃度的肽處理之 後的細胞生長抑制率； 圖9顯示小鼠巨噬細胞RAW 264.7經不同濃度的肽處 理之後所測得的NO生成抑制率； 圖10顯示使用胃蛋白酶來對小球藻廢棄物進行水解所 得到的胃蛋白晦水解物的SDS-PAGE電泳圖，其中徑M表 不蛋白質標記（Protein marker);徑S表示未經水解的小球藻 廢棄物；以及徑p表示胃蛋白酶水解物； 圖U顯示使用胃蛋白酶來對小球藻廢棄物進行水解所 得到的胃蛋白酶水解物的ABTS自由基清除活性，其中s 表不未經水解的小球藻廢棄物；以及P表示胃蛋白酶水解 物； 圖12顯示本案實施例7當中所進行的凝膠過濾層析的 洗提圖形，其中G1與G2分別表示所得到的分離部分；以 及箭頭是示意分離部分的範圍； 圖13顯示本案實施例7當中所進行的逆相高效能液相 層析的洗提圖形，其中P2表示所得到的分離部分； 圖14顯示本發明的肽、BHT、Tr〇l〇x以及抗壞血酸的 ABTS自由基清除活性； 圖15顯示本發明的肽、Trol〇x以及抗壞血酸的DPPH 自由基清除活性； 圖16A與圖16B分別顯示本發明的肽、BHT以及 ΤΓ〇1〇Χ的羥基自由基清除活性以及超氧自由基清除活性； 53 201043239 圖17顯示本發明的肽、Trolox、BHT以及抗壞血酸在 ORAC分析中所得到的螢光衰退曲線； 圖18顯示不同濃度的肽對於羥基自由基-誘發的質體 DNA傷害的防護效用，其中OC DNA表示：呈現開環（open circular, OC)構形的DNA; SC DNA表示：呈現超螺旋 (super coil, SC)構形的DNA;徑1是不作任何處理的質體 pET-28a (正常對照組）；徑2是使用FeS04與H202來誘發氧 化性傷害的質體pET-28a (正對照組）；以及徑3至徑5是分 別以濃度為84.9、42.4以及10.6 μΜ的肽來處理經羥基自 由基-誘發氧化性傷害的質體pET-28a ; 圖19A至圖19C分別顯示未經任何處理、經H2〇2處理 以及經H2〇2與本發明的肽處理的AGS細胞的型態；以及 圖20顯示人類胃腺癌細胞AGS經不同濃度的肽處理之 後的細胞存活率。 【主要元件符號說明】 (無） 54 201043239 序列表 <110> 國立中興大學 <120> —種經分離的肽及其製備方法與應用 <130> NCHU <160〉 4 <170> Patentln version 3.4The lyophilized fraction F2 obtained in item B above was dissolved in 5 mL of deionized water and condensed according to the following operating conditions. Glue Filtration Chromatography: The column was buffered with 20 mM Tris-HCl. Sephacryl S-100 high HR column (length: 2_6 cm X 70 cm) pre-equilibrated in solution (pH 7_8); eluent was 20 mM Tris-HCl buffer solution (pH 7.8); flow rate 〇 was controlled to 1.5 mL/ Min ; The detection wavelength is set to 220 nm. At the time of elution, each 6 mL of the eluate was collected in a test tube. The eluate corresponding to each peak was collected based on the obtained elution pattern, and then the antioxidant activity was measured in accordance with the method described in "B, Trolox equivalent antioxidant capacity analysis" of "General Experimental Method" above. The experimental results revealed that two peaks were observed in the elution pattern of the gel filtration chromatography. Separate portions corresponding to the peaks are separately collected to obtain separated portions G1 and G2 (see Fig. 12). The antimony antioxidant activity of the separated fractions G1 and G2 was measured, and it was found that the lower molecular weight fraction G2 had a higher antioxidant activity than that of the fraction G1 (about 2.5 times) ( The data is not shown). Therefore, the separated portion G2 was freeze-dried for the following analysis. D. Anion exchange chromatography: The lyophilized fraction G2 obtained in the above item C is dissolved in 5 mL of deionized water, and the method described in "C, Anion Exchange Chromatography" of Example 2 above is referred to. Anion exchange chromatography was performed, except that 35 201043239 was that the detection wavelength was set to 220 nm. The eluate corresponding to each peak was collected based on the obtained elution pattern, and then the antioxidant activity was measured in accordance with the method described in "B, Trolox equivalent antioxidant capacity analysis" of "General Experimental Method" above. As a result of the experiment, it was found that the separated fraction collected when the NaCl concentration of the eluate was 0 具有 had a high antioxidant activity (data not shown). Therefore, the separated fraction having higher antioxidant activity was collected, which was then freeze-dried for the following analysis. Ε, reverse phase high performance liquid chromatography: The lyophilized fraction obtained in item D above is dissolved in 1 mL of deionized water and subjected to reverse phase high performance liquid chromatography according to the following operating conditions. The instrument model is Agilent 1100 (Agilent Technologies Inc. CA, USA); the analytical column is an Inertsil ODS-3 C18 semi-preparative column (10 mm x 250 mm); the eluent has an acetonitrile gradient (l5 to 35%, 30 minutes) of an aqueous solution containing 0.1% TFA; the flow rate was controlled to 2 mL/min; the detection wavelength was set to 220 nm. The eluate corresponding to each peak was collected based on the obtained elution pattern, and then the antioxidant activity was measured in accordance with the method described in "B, Trolox equivalent antioxidant capacity analysis" of "General Experimental Method" above. As a result of the experiment, it was found that a peak was detected when the residence time was about 23 minutes (see Fig. 13), and the eluted material corresponding to the peak (i.e., the separated portion P2) had antioxidant activity. The isolated fraction P2 was freeze-dried to obtain a peptide having antioxidant activity. 36 201043239 The obtained peptide having antioxidant activity was confirmed by using an Agilent 6510 Q-TOF mass spectrometer (Agilent Technologies Inc. CA, USA) to confirm the purity. The results showed that the peptide obtained in this experiment had a purity greater than 98%. Therefore, the obtained peptide was subjected to the following amino acid sequence analysis. F, Amino Acid Sequence Analysis: The peptides obtained in item E above were subjected to amino acid sequence analysis by using an Applied Biosystems Procise 494 Protein Sequencer and referring to the 〇 operating instructions provided by the manufacturer. The analysis results showed that the amino acid sequence of the peptide having antioxidant activity obtained in the present example was the same as that of the peptide having ACE inhibitory activity obtained in the above Example 2. Further, 'Applicants have verified that the peptide obtained in this example does have antioxidant activity, and a peptide having an amino acid sequence as shown in SEQ ID NO: 1 is synthesized' and according to the above "general experimental method, , "B, Trolox equivalent antioxidant capacity analysis," the method described to determine antioxidant activity. The results showed that the synthesized peptide also had antioxidant activity as the peptide obtained in the present example (data not shown). Example 8. Antioxidant effect of a peptone hydrolysate-derived peptide isolated from chlorella waste to understand peptides of pepsin hydrolysate isolated from chlorella waste according to the present invention [Including: ABTS free radicals, DPPH free radicals, peroxyxy radicals (per〇xyi radicais, hydroxyl radicals, and superxx radicals)] The peptide obtained in Example 7 was subjected to the following experiment of 37 201043239. Experimental method '· A, Trolox equivalent anti-gasification capacity analysis: 1. Antioxidant solution and preparation of ABTS solution: In this experiment, the peptide, Trolox and ascorbic acid obtained in the above Example 7 were respectively Formulated in deionized water, BHT was formulated in 1% DMSO to obtain antioxidant solutions with different concentrations. Further, the ABTS solution was prepared by the method described in M.J.T.J. Arts d a/. (2004) (same as above). 2. Experimental procedure: The TEAC analysis of each antioxidant solution was carried out by referring to the method described in "B, Trolox equivalent antioxidant capacity analysis" of the "general experimental method" above, and the antioxidant concentration in the mixture was Shown in Table 3 below. Table 3. Antioxidant concentration Antioxidant concentration (μΜ) Peptide 3.56 ' 7.12 ' 14.24 ' 28.48 BHT 3.56, 7.12, 14.24, 28.48 Trolox 11.90 ' 23.81 ' 35.71 Ascorbic acid 7.44 ' 14.87 ' 29.75 In addition, these four antioxidants The concentration of the ABTS free radical (IC5〇) of 50% of the substance was calculated. The experimental data obtained were analyzed in accordance with the method described in "C, Statistical Analysis" of the "general experimental method" above. 38 201043239 B 'DPPH radicals scavenging activity assay: 1. Preparation of antioxidant solution and DPPH solution: In this experiment, the peptide obtained in the above Example 7 and ascorbic acid were separately prepared. In deionized water, BHT and Trolox were separately formulated in 1% DMSO and absolute alcohol to obtain antioxidant solutions with different concentrations. In addition, the DPPH solution is prepared by the method described in SJ Huang and JL Mau (2006), 5W. Tec/mo/., 39:707-716. 2. Experimental procedure: DPPH free radicals of various antioxidant solutions The scavenging activity analysis was carried out in accordance with the method described in SJ Huang and J_L. Mau (2006) (supra). Briefly, different concentrations of antioxidant solution (4 mL) were separately mixed with DPPH solution (1 mL) to form a mixture containing different concentrations of antioxidants (see Table 4)*. Thereafter, the resulting mixture was left to stand in the dark for a period of 40 minutes, and then the absorbance (OD517) was measured at a wavelength of 517 nm. In addition, the control group was used in the preparation of the antioxidant solution. A solvent to replace the antioxidant solution. Table 4. Antioxidant concentration Antioxidant concentration (μΜ) Peptides 29.9083, 59.8167, 119.6 BHT 454.55, 909.09, 1818.18 Trolox 11.99, 23.97, 31.96, 95.88 Ascorbic acid 10.68, 21.35, 42.70, 85.41 39 201043239 Individual antioxidant solutions The DPPH free radical scavenging activity was calculated by substituting the measured OD517 value into the following formula: A(%) = [(BC)/B]xl00 where: A = DPPH free radical scavenging activity B = control group The OD517 value of the OD517 value of the antioxidant solution was determined by the addition of 50% of the DPPH free radical concentration (IC5G) of the four antioxidants. The experimental data obtained were analyzed in accordance with the method described in "C, Statistical Analysis" of the "general experimental method" above. C. Hydroxyl radicals scavenging ability assay: 1. Preparation of antioxidant solution and reactant solution: In this experiment, the peptide obtained in the above Example 7 and Trolox were separately formulated. Deionized water was used while BHT was formulated in 1% DMSO to obtain antioxidant solutions with different concentrations. In addition, the reactant solution (containing 1.0 mL of 3 μΜ IBG, 0.1 mL of 1.0 mM FeS04, 1.6 mL of 3% H 2 〇 2 and 0.05 mL of 10 mM EDTA) is referred to CH Tsai < 2 Plant (2001), / · Prepared by the method described in JgTic· Food C/iem·, 49: 2137-2141. 2. Experimental procedure: The analysis of the radical radical scavenging ability of each antioxidant solution was carried out by the method described in C.H. Tsai ei a/. (2001) (same as above). Briefly, different concentrations of antioxidant solution (10 μιη) were separately added to the above reactant solution to form a mixed 40 201043239 compound containing different concentrations of antioxidants (see Table 5). High Sensitivity> (BJL-ultraweak chemiluninescence analyser, Jye Horn Co. Taiwan) to detect changes in cheilininescence (CL). In addition, the control group was prepared. The antioxidant solution was used to replace the antioxidant solution. Table 5. Antioxidant concentration Antioxidant concentration (μΜ) Peptides 1.99, 3.99, 5.97, 7.96, 9.95 ΒΗΤ 16.53, 33.06, 49.59, 66.12 Trolox 1.45, 2.91, 4.36, 5.81 The radical radical scavenging activity of each antioxidant solution was calculated by substituting the measured chemical counts (CL counts) into the following formula: A (%) = [( BC)/B]xl00 where: A = hydroxyl radical scavenging activity B = chemical fluorescence number of the control group 化学 chemical fluorescence number of the antioxidant solution, in addition, the removal of 50% of these three antioxidants is free Thick The degree (IC5〇) was calculated. The experimental data obtained were analyzed according to the method described in “C. Statistical Analysis” of “General Experimental Methods” above. D. Superoxide radicals analysis Scavenging ability assay) ' 1. Preparation of antioxidant solution and reactant solution: In this experiment, the peptide obtained in Example 7 above and Trolox were separately prepared in deionized water, and BHT was formulated in 1% DMSO. Medium 41 201043239 to obtain antioxidant solutions with different concentrations. In addition 'reactant solution [containing 1.0 mL of 2. mM luciferin (lucigenin), 0. 〇 5 mL of 1.0 Μ arginine, 〇〇 5 machine i 4 _ pyruvic aldehyde and 1.0 mL of PBS buffer (ΡΗ 7.4)] is referred to c H Tsai ei fl /. (2003), / · Food CAem., 51: 58-62 Preparation 2. Experimental procedure: The analysis of superoxide radical scavenging activity of each antioxidant solution was carried out by referring to the method described in C.H_ Tsai ei a/. (2003) (same as above). In short, 'will be different Concentration of antioxidant solution (10 added to the above Reactant solution to form a mixture containing various concentrations of antioxidants (see Table 6), which in turn varies with the use of a detector of a high sensitivity BJL-Ultraweak chemiluminescence analyzer to detect the chemiluminescence. In addition, the control group replaced the antioxidant solution with the solvent used in the preparation of the antioxidant solution. Table 6 · Antioxidant concentration Antioxidant concentration (μΜ) Peptides 3.38, 6.75, 10.13, 13.50, 16.88 BHT 44.35, 88.69, 133.04, 177.38, 221.73 Trolox 19.49, 38.98, 58.47 The superoxide radical scavenging activity of each antioxidant solution was calculated by substituting the measured chemical fluorescence number into the following formula: A(%)= [(BC)/B]xl〇〇 where: A=superoxide radical scavenging activity 42 201043239 B=chemical fluorescence number of control group C=chemical fluorescence number of antioxidant solution In addition, these three kinds of antioxidants The concentration of 50% superoxide radicals (IC5〇) was calculated. The experimental data obtained were analyzed in accordance with the "general experimental method," "C, statistical analysis," the method described above. E, oxygen free radical absorption capacity analysis [〇xygen radical absorbance capacity (ORAC) Assay]: 1. Preparation of antioxidant solution, luciferin solution and aaph solution: 〇 In this experiment, obtained in Example 7 above The peptide, BHT, ascorbic acid and Trolox were each formulated in 75 mM phosphate buffered saline (PBS) to obtain an antioxidant solution. Trolox is used as a standard. In addition, a 96 nM luciferin solution and a 320 mM AAPH solution were obtained by fitting an appropriate amount of luciferin and AAPH in 75 mM PBS, respectively. ' 2. Experimental procedure: The ORAC analysis of each antioxidant solution is based on the method described in D. Alberto w α/. (2004), J. JgWc. Food CAem·, 52:48-54 and slightly modified. Later. Briefly, 20 pL of the antioxidant solution and 150 pL of the luciferin solution were added to the well of the microplate, followed by pre-incubation at 37 °C for 5 minutes. Thereafter, a 30 μί AAPH solution was added to initiate the reaction, and the concentration of the antioxidant in the resulting mixture was shown in Table 7 below. 43 201043239 Table 7. Antioxidant Concentrations Antioxidant Concentration (μΜ) Peptide 3.7 '7.5 BHT 29.9 Trolox 10 Ascorbic Acid 31.2 Next, immediately place the mini-disc in the reader for one minute Fluorescence values were recorded at intervals for a total of 130 minutes, thereby obtaining fluorescene decay curves for each of the antioxidant solutions. In addition, the control group was replaced with 75 mM PBS to replace the antioxidant solution. .. A, Trolox equivalent antioxidant capacity analysis: Figure 14 shows the ABTS free radical scavenging activity of the peptide, BHT, Trolox and ascorbic acid of the present invention. As can be seen from Fig. 14, the four antioxidants were dose-dependent for the scavenging activity of ABTS radicals. In addition, in terms of the ability to scavenge ABTS free radicals, the highest is the peptide of the present invention (IC50 value is 9·8±0·5 μΜ), followed by ascorbic acid (IC5〇 value is 30.0±0·8 μΜ) and Trolox (IC5〇 value is 32·5±1·3 μΜ)' and the synthesized antioxidant BHT is the lowest (IC5〇 value is 57.4±2·3 μΜ). Β, DPPH free radical scavenging activity analysis: Figure 15 shows the DPPH free radical scavenging activity of the peptide, Trolox and ascorbic acid of the present invention. As can be seen from Fig. 15, the peptide of the present invention has a dose-dependent activity for DPPH free radical scavenging at a concentration of 29.9083 and 59.8167 μM. In addition, in terms of the ability to scavenge DPPH free radicals, the highest was Trolox (IC5〇 value was 23.0±1.8 μΜ), followed by ascorbic acid (IC5〇 value was 44.0 μΜ) and the peptide of the present invention (IC5〇 value was 58.0±) 1·2 μΜ), and the synthesized antioxidant BHT is the lowest (IC50 value is 968·3±2.6 μΜ) (not shown). In addition, a similar previous study showed that peptides derived from bullfrog skin have an IC5〇 value of 16.1 μΜ (ZJ Qian α/. (2008a), e/oresowr. JlecAwo/., 99:1690-1698) The peptide derived from casein has an IC50 value of 98 μΜ (K. Suetsuna α/. (2000), /· iVwiW. 5/ocAem., 11: 128-131). Comparison of the peptides of the present invention with such conventional peptides revealed that the peptides of the present invention have moderate resistance to DPPH free radicals. C. Analysis of the radical scavenging ability of the radical: Fig. 16A shows the hydroxyl radical scavenging activity of the peptide, BHT and Trolox of the present invention. As can be seen from Fig. 16A, the scavenging activities of the three antioxidants for the hydroxyl radical were dose-dependent. In addition, in terms of the ability to scavenge radicals, the highest is Trolox (IC5〇 value is 2.0±0.3 μΜ), followed by the peptide of the present invention (IC5〇 value is 8.3±0.15 μΜ), and BHT (IC5〇) The value is 44.4 ± 1.6 μΜ) which is the lowest. D. Analysis of supergas radical scavenging ability: Fig. 16A shows the superoxide free radical scavenging activities of the peptide, guanidine and Trolox of the present invention. As seen from Fig. 16B, the superoxide radical scavenging activity of the peptide of the present invention increases sharply with an increase in concentration. In addition, in terms of the ability to scavenge superoxide radicals, the highest is the peptide of the invention (IC5〇 value is 7.5 45 201043239 ±0.12 μΜ), followed by Trolox (IC5〇 value is 24.8±0·4 μΜ) , and ΒΗΤ (IC5〇 value is 218_1±2.1 μΜ) is the lowest. In addition, a similar previous study showed that the peptide derived from the cool protein has an IC50 value of 79.2 μΜ (K. Suetsuna et al. (2000), supra), and the peptide derived from the oyster protein hydrolysate has a 1C5Q value of 78.971111. ^(2:.)· Qian a/. (2008b), price 0asowr· rec/iwo/., 99:3365-3371), and 2 peptides derived from muscle protein hydrolysate of giant squid The IC5 values are 669.34 and 573.83 μΜ, respectively (N. Rajapakse ei a/. (2005), same as above). Comparing the peptide of the present invention with these conventional peptides, it was found that the peptide of the present invention has the lowest value of 1C5() (i.e., has a good ability to scavenge superoxide radicals at a low concentration). E. Oxygen Radical Absorption Capacity Analysis: Figure 17 shows the fluorescence decay curves obtained for the peptides of the present invention, Tr〇l〇x, BHT, and ascorbic acid in ORAC analysis. As can be seen from Figure 17, the peptide of the present invention has a dose-dependent increase in inhibition of fluorescein decline. In addition, in terms of the ability against peroxy1 radicals, the highest is the peptide of the present invention, followed by Trolox, followed by ascorbic acid, and BHT is the lowest. Peroxyl radicals are formed in lipid peroxidation 'so' Applicants' inference: the peptides of the invention can terminate the free radical bond reaction of lipid peroxidation by scavenging peroxy radicals (free radicals) Chain reaction). Example 9 Evaluation of the protective effect of peptides having antioxidant activity on hydroxyl radical-induced plasmid DNA damage 46 201043239 To understand the separation of chlorella waste according to the present invention The peptide pepsin hydrolysate peptide has a protective effect against DNA damage induced by hydroxyl radicals, and the applicant uses the peptide obtained in Example 7 to carry out the following experiment. The actual method is as follows: • This experiment is a reference to ZJ Qian ei The method described in a/. (2008b) (same as above) is slightly modified and then carried out. Briefly, an appropriate amount of the peptide obtained in the above Example 7 was dissolved in 1 mL of deionized water to form a peptide solution having a concentration of 10.6, 42.4 and 84.9 μM. In addition, the control group was replaced with deionized water to replace the peptide solution. 4 μί of the peptide solution was thoroughly mixed with 8 pL of plastid pET-28a, 3 pL of 2 mM FeS〇4 and 4 pL of 0.06 mM H 2 O 2 to form a reaction mixture, followed by pre-incubation at 37 ° C for 30 minutes. . Thereafter, it was analyzed by 1% agarose gel electrophoresis, followed by staining with SYBR Green, and then observed under ultraviolet light and photographed. The normal control is the plastid O pET-28a without any treatment, while the positive control is the plastid pET-28a treated with FeS〇4 and Η2〇2. Results: Figure 18 is an electrophoresis film showing the results of agarose gel electrophoresis analysis of hydroxyplast-induced oxidative damage plastid DNA treated with different concentrations of peptides. Wherein 〇C DNA means: DNA showing an open circular (OC) configuration; SC DNA means: 47 201043239 DNA exhibiting a super coil (SC) configuration, plastid pET with a diameter of 1 疋 without any treatment 28a (normal control group); 仫2疋 using FeS04 and h2〇2 to induce oxidative damage to the plastid ρΕτ· 28a (positive control group); and diameter 3 ligament 5 at concentrations of 84 9 , 42.4 and 10.6 μΜ (4) To treat the plastid PET_28a, which is caused by secret free radical-induced oxidative damage. The experimental results show that nucleotide free radicals induce oxidative damage to transform plastid DNA from supercoiled to open-loop configuration (see Diameter 2), however, the peptides of the present invention are effective in preventing oxidative damage. The resulting DNA configuration changes and the protective effect increases with increasing concentration (see Run 3 to Path 5). Example 1G. The runt toxicity analysis of peptides having anti-sputum activity and the evaluation of the protective effect of human gastric adenocarcinoma ags with H2〇2_induced cell damage is to understand the basis The protective effect of the peptide of the pepsin hydrolyzate of the invention isolated from chlorella waste on the cell damage induced by 4〇2 and the toxic effect on cells, the applicant uses the peptide obtained in Example 7 to carry out the following experiment of. Experimental methods: A. Evaluation of the protective effect of the peptide of the present invention on 1|2〇2· induced cell injury: AGS cells were seeded into 96-well plates containing RPMI medium (containing 1% fetal calf blood 'months) (4 χ 1 〇 3 cells/well), and then cultured at 37 〇 c for 24 hours. Next, an appropriate amount of the peptide solution (the peptide obtained in the above Example 7 was added to deionized water) was added to a final concentration of 〇 2, 48 201043239 0.04, 0.08, and 0.15 mM, respectively, followed by incubation for 2 hours. Thereafter, 20 pL of H2〇2 (final concentration of 0.03 mM) was added and cultured for 2 hours. At the 2nd hour of H2〇2 treatment, the cell type of the culture was observed by a phase-contrast microscope (Nikon). The normal control was AGS cells without any treatment, while the negative control was AGS cells treated with 0.03 mM H2〇2. Next, MTT (20 μί/well) was added to each well and cultured for 4 hours. Thereafter, 200 μL of 100% DMSO was added to each well to dissolve the ΜΤΤ-methyl hydrazine. The absorbance of each well (〇D57G) was read at a wavelength of 570 nm using a microtiter plate reader. Cell viability was calculated by substituting the measured 〇D57〇 value into the following formula: A(%) = (C/B)xl00 where: A = cell viability B = OD57 of the normal control group The numerical data of the 'C = OD570 value of the experimental group or the negative control group 〇 was obtained by the method described in "C, Statistical Analysis" of "General Experimental Method" above. B. Cytotoxicity test of the peptide of the present invention on human normal lung cell WI-38 WI-38 cells were inoculated into a 96-well plate (3 x 1003 cells/well) containing DMEM medium (containing 10% fetal calf serum). The cultivation was then carried out at 37 ° C for 24 hours. Next, an appropriate amount of peptide solution (the peptide obtained in Example 7 above was formulated in deionized water) was added to each well to a final concentration of 49 201043239, which was 0.02, 〇_〇4, 0.08, and 〇_15 mM, It was cultured for 72 hours. Cells not treated with peptide were used as a control group. Next, MTT (20 gL/well) was added to each well and cultured for 4 hours. Thereafter, 200 μL of 100% DMSO was added to each well to dissolve the ΜΤΤ-甲臜. A microtiter plate reader was used at a wavelength of 570 nm to read the absorbance of each well (〇D57〇). Cell viability was calculated by substituting the measured 〇D57〇 value into the following formula: A(%) = (C/B)xl00 where: A=cell survival rate OD57〇 value of control group C= The experimental data obtained from the 〇D57〇 value of the experimental group were analyzed according to the method described in “C, Statistical Analysis” of the above-mentioned “general experimental method.” Submissions.· FIGS. 19A to 19C respectively show no The morphology of ags cells treated with any treatment, treated with h2〇2 and treated with H2〇2 and the peptide of the present invention. As can be seen from Figures 19A to 19C, untreated AGS cells have intact cell types and undergo H2. The cells treated with 〇2 showed obvious bubbles. However, before the AGS cells were treated with H2〇2, if the peptide of the present invention was previously cultured for 2 hours, the cell damage induced by Η" This result is shown to be significantly reduced. This result shows that the peptide of the present invention has a protective effect against h2〇2-induced cell damage. Figure 20 shows the cell survival rate of human gastric adenocarcinoma AGS after treatment with different concentrations of peptide. With ha induced cells The survival rate of AGS cells with injury 50 201043239 is only about 20%. However, AGS cells previously cultured with the peptide of the present invention still have superior survival rate after H202-induced cell injury, and their survival rate will follow The increase in peptide concentration increased. This result shows that peptides isolated from pepsin hydrolysate of chlorella waste have a dose-dependent protective effect on cell damage induced by H202. To investigate the cytotoxicity of the peptide of the present invention to human normal lung cell WI-38, the experimental results show that no growth inhibition is observed after wi-38 cells are treated with different concentrations of ruthenium peptide, and thus the shape of the present invention is human. The normal lung cell WI-3 8 has no cytotoxicity effect (data not shown). From the above experimental results, it is understood that the peptide of the pepsin hydrolyzate of the present invention isolated from chlorella waste has excellent resistance. Oxidative effect. Therefore, the peptide of the present invention is expected to be useful for the preparation of a disease for preventing damage caused by oxidative damage [such as an artery Pharmaceutical or dietary supplements of atheroscierosis, coronary heart disease, cancer, Alzheimer's disease, aging, gastric ulcer, etc. All references cited in this prospectus And the patent documents are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the the the the the the the the the the the the the the Many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only by the scope of the appended claims. 51 201043239 [Simple description of the schema] Figure 1A and Figure 1B show the protein yield measured by chlorella waste after hydrolysis of different eggs and ACE inhibited live white peony, 1 king, where "less" means ρ <0·05; Fig. 2 shows the ACE inhibitory activity of the isolated fraction obtained by the separation of the gastric protein plum hydrolysate of the chlorella waste by the eight-stage separation of sulfuric acid, and the ratio of A4 to A4 respectively corresponds to 〇~2〇%, 2 〇~4〇%, 4〇~%^A1 60~80% of the separated portion of the sulfuric acid recording saturation; and FIG. 3 shows the separated portions B1 and B2 obtained in the gel filtration chromatography of Example 2 of the present invention Figure 4A shows the elution pattern of the anion elution carried out in Example 2 of the present case, where C1 and C2 respectively represent the separated portions obtained: the arrow is the range indicating the separated portion; and the broken line indicates the Ν& α gradient. Fig. 4B shows the ACE inhibitory activity of the separated fractions C1 and C2 in the anion exchange chromatography of Example 2 of the present invention; Fig. 5A and Fig. 5B show the use of mensii ODS-3 C18 half in the present embodiment 2, respectively. Prepare grade column and use postal The C8 column is used for the elution pattern of reverse phase high performance liquid chromatography, wherein (1) and Η represent the separated parts, respectively; and the arrow is the range indicating the separation; Figure 6 shows the separation from chlorella waste. The ACE inhibitory activity of the peptides of the gastric protein hydrolysate at different concentrations; Figure Illustrator shows the inhibition of ACE inhibition at different concentrations of the gastric protein peak hydrolysate from the chlorella waste Figure 52 201043239 Figure 8 shows the cell growth inhibition rate of human gastric adenocarcinoma AGS after treatment with different concentrations of peptide; Figure 9 shows the NO inhibition inhibition rate of mouse macrophage RAW 264.7 after treatment with different concentrations of peptide; Figure 10 shows an SDS-PAGE electrophoresis pattern of pepsin hydrolysate obtained by hydrolyzing chlorella waste using pepsin, wherein the diameter M indicates a protein marker (Protein marker); the diameter S indicates a small amount of unhydrolyzed Chlorella waste; and the diameter p represents pepsin hydrolysate; Figure U shows ABTS free radical scavenging of pepsin hydrolysate obtained by hydrolysis of chlorella waste using pepsin , wherein s represents unhydrolyzed chlorella waste; and P represents pepsin hydrolysate; FIG. 12 shows a elution pattern of gel filtration chromatography performed in Example 7 of the present invention, wherein G1 and G2 are respectively The resulting separated portion is indicated; and the arrow is the range indicating the separated portion; FIG. 13 shows the elution pattern of the reverse phase high performance liquid chromatography performed in Example 7 of the present invention, wherein P2 represents the obtained separated portion; 14 shows the ABTS radical scavenging activity of the peptide, BHT, Tr〇l〇x and ascorbic acid of the present invention; Fig. 15 shows the DPPH radical scavenging activity of the peptide of the present invention, Trol〇x and ascorbic acid; Fig. 16A and Fig. 16B show Hydroxyl radical scavenging activity and superoxide radical scavenging activity of the peptide, BHT and ΤΓ〇1〇Χ of the present invention; 53 201043239 Figure 17 shows the fluorescence obtained by the peptide of the present invention, Trolox, BHT and ascorbic acid in ORAC analysis Decline curve; Figure 18 shows the protective effect of different concentrations of peptide on hydroxyl radical-induced plastid DNA damage, where OC DNA means: open circular (OC) Configuration DNA; SC DNA: DNA showing super coil (SC) configuration; diameter 1 is plastid pET-28a (normal control) without any treatment; diameter 2 is using FeS04 and H202 The plastid pET-28a (positive control group) which induces oxidative damage; and the plastid pET which treated the hydroxyl radical-induced oxidative damage by peptides of 84.9, 42.4 and 10.6 μΜ, respectively, in diameters 3 to 5 -28a; Figures 19A to 19C show the types of AGS cells treated with H2〇2 and treated with H2〇2 and the peptide of the present invention, respectively, without any treatment; and Figure 20 shows different concentrations of human gastric adenocarcinoma AGS. Cell viability after peptide treatment. [Description of main component symbols] (none) 54 201043239 Sequence Listing <110> National Chung Hsing University <120> - Isolated peptide and preparation method and application <130> NCHU <160> 4 <170> Patentln version 3.4

<210> 1 <211> 11 <212> PRT <213> 人工的 <220><210> 1 <211> 11 <212> PRT <213> Manual <220>

<223> 分離自小球藻廢棄物的胃蛋白酶水解物 <400〉 1<223> Pepsin hydrolysate isolated from chlorella waste <400〉 1

Val Glu Cys Tyr Gly Pro Asn Arg Pro Gin Phe <210> 2 <211> 6 10 201043239 <212〉 PRT <213> 人工的 <220> <223〉合成肽PEP1 <400> 22, lt; 211 > 6 10 201043239 <212>

Pro Asn Arg Pro Gin Phe 1 5 <210> 3 <211> 6 <212> PRT <213> 人工的 <220〉 <223> 合成肽PEP2 <400〉 3 Val Glu .Cys Tyr Gly 1 5 <210> 4 <211〉 6 2 201043239 <212> PRT <213> 人工的 <220> <223〉合成肽PEP3 <400> 4<210> 3 &lt Gly 1 5 <210> 4 <211> 6 2 201043239 <212> PRT <213> Artificial <220><223>synthetic peptide PEP3 <400>

Cys Tyr Gly Pro Asn Arg o 5Cys Tyr Gly Pro Asn Arg o 5

Claims (1)

201043239 七、申請專利範圍： 其具有一如序列辨識編號：1所示的 2·如申請專利範圍第1項的肽 之方法而被製得： 提供種屬於—小球藻屬藻類（algae of CA/㈣"α genus)的藻類材料； 將該藻類材料推# ..201043239 VII. Patent application scope: It has the same method as the peptide identification number: 1 according to the sequence identification number: 1. The peptide is obtained according to the method of the peptide of the first paragraph of the patent: The species is provided - algae of CA / (d) "α genus) algae material; push the algae material to #.. 造仃一使用一蛋白酶的水解處理，藉 此而得到一水解物；以及 將3玄水解物進行一純化處理，藉此而得到該肽。 3‘ ㈣_第2項的月太，其中在該方法中，該小球 _類是選自於下列所構成的群組：小球_㈣"《 VM/gaW5)、蛋白核小球藻（⑶州//α w酬㈣⑽）、橢圓 小球藻(〜//ae//—，·“)、微小小球藻 觀――、耐熱性小球藻__觀），以 及它們的組合。The peptide is hydrolyzed by a protease to thereby obtain a hydrolyzate; and the 3x hydrolyzate is subjected to a purification treatment, thereby obtaining the peptide. 3' (4) _ Item 2 of the month too, wherein in the method, the ball _ class is selected from the group consisting of: small ball _ (four) " "VM / gaW5", protein chlorella ( (3) State / / α w reward (four) (10)), ellipsoids (~ / / ae / / -, · "), micro-chlorella view -, heat-resistant chlorella __ view), and combinations thereof. 1. 一種經分離的肽 胺基酸序列。 它是藉由一包含下列步驟 4.如申-月專利範圍帛3項的肽，其中在該方法中該小球 藻屬藻類是小球藻（c/l/〇re//fl vm/_㈨。 5·如申請專利_ 2項的肽，其中在該方法中，該蕩類 材料是天然的材料。 6·如中請專利範圍帛2項的肽，其中在該方法中，該藻類 材料在被進行水解之前，有先經過一加工處理。 7.如申請專利範圍“項的肽，其中該加工處理是選自於 下列所構成的群組：水萃取處理、酵素水解處理、熱處 55 201043239 、南溫高壓處理、高壓均質處理、酸處理 及冷柬解象處理，以及它們的組合。 良 8.如申請專利範圍第2項的狀，其t在該方法_，該蛋白 酶是選自於下列所構成的群組··胃蛋白酶、驗蛋白酶、 木瓜蛋白酶、A型風味蛋㈣，以及它們的組合。 •如申凊專利範圍第8項的肽，其中在該方法中，該蛋白 酶是胃蛋白轉。 !〇.如申，專利範圍第2項的肽，其中在該方法中，該純化 處理疋選自於下列所構成的群組：硫酸胺分級分離處理 、凝膠過濾、層析、離子交換層析、逆相層析、疏水性交 互作用層析，以及它們的組合。 11. 一種具有血管緊縮素！轉化酵素抑制活性的藥學組成物 ’其包含有-如申請專利範圍第】至1〇項中任一項的肽 〇 12. —種具有抗氧化活性的藥學組成物，其包含有一如申請 專利範圍第1至10項中任一項的肽。 13. —種用於抑制癌細胞增生的藥學組成物其包含有一如 申請專利範圍第1至10項中任一項的肽。 14. 如申明專利範圍第丨3項的藥學組成物其中該癌細胞是 胃腺癌細胞。 1 5. —種具有抗發炎活性的藥學組成物，其包含有一如申請 專利範圍第1至10項中任一項的肽。 16 · —種食品產品，其包含有一如申請專利範圍第丄至丨〇項 中任一項的肽。 56 Ο ❹ 201043239 17.—種用以製備一具右一 L + 、 如序列辨識編號：1所示的胺基 酸序列的肽的方法，其包括： 提供一 厲 ;—小球藻屬藻類（algae of genus)的藻類材料； 將該藻類材料谁并,.m 叶進仃一使用一蛋白酶的水解處理，藉 此而得到一水解物；以及 將〜水解物進行一純化處理，藉此而得到該月太。 A如申請專利範圍第17項的方法，其中該小球藻屬藻類是 、自於下歹j所構成的群組：小球藻（㈤㈣""咖、 蛋白核J球藻、橢圓小球藻 (Chlorella eilips〇ide幻、敬]、小來薄仰ι〇_α "咖;ηα)、耐熱性小球藻(⑶㈣"β，以 及它們的組合。 19. 如申胡專利範圍第i 8項的方法其中該小球藻屬藻類是 々、珠蕩（Chlorella vulgaris)。 20. 如申請專利範圍第17項的方法，其中該藻類材料是天然 的材料。 21. 如申請專利範圍第17項的方法，其中該藻類材料在被進 行水解之前，有先經過一加工處理。 22. 如申請專利範圍第21項的方法，其中該加工處理是選自 於下列所構成的群組：水萃取處理、酵素水解處理、熱 處理、高溫高壓處理、高壓均質處理、酸處理、驗處理 以及冷凍解凍處理，以及它們的組合。 23. 如申請專利範圍第17項的方法，其中該蛋白皞是選自於 57 201043239 下列所構成的群組：胃蛋白酶、鹼蛋白晦、 Λ _ϊγ丨 小"瓜·蛋白酸_ ' A !風味蛋白酶，以及它們的組合。 24 =申請專利範圍第23項的方法，纟中該蛋白梅是胃蛋白 25.=申請專利範圍第17項的方法，其中該純化處理是選自 2列所構成的群組：硫酸胺分級分離處理、凝膠過遽 斤、離子交換層析、读相恳把 ,P, „ ^ 逆相層析、疏水性交互作用層析 M及它們的組合。 581. An isolated peptide amino acid sequence. It is a peptide comprising the following steps 4. For example, the patent scope of the patent application, in which the chlorella algae is chlorella (c/l/〇re//fl vm/_(9) 5. The peptide of claim 2, wherein in the method, the smear material is a natural material. 6. The peptide of the patent scope 帛 2, wherein in the method, the algae material is Before being hydrolyzed, it is subjected to a processing treatment. 7. The peptide of the item of the patent application, wherein the processing is selected from the group consisting of: water extraction treatment, enzyme hydrolysis treatment, heat treatment 55 201043239 , South temperature high pressure treatment, high pressure homogenization treatment, acid treatment and cold treatment, and combinations thereof. Good 8. As in the scope of claim 2, the t is in the method _, the protease is selected from a group consisting of pepsin, protease, papain, type A flavored egg (four), and combinations thereof. The peptide of claim 8, wherein in the method, the protease is a stomach Protein transfer. !〇.如申, patent scope 2 a peptide, wherein in the method, the purification treatment is selected from the group consisting of: ammonium sulfate fractionation treatment, gel filtration, chromatography, ion exchange chromatography, reverse phase chromatography, hydrophobic interaction Chromatography, and a combination thereof 11. A pharmaceutical composition having an angiotensin! transforming enzyme inhibitory activity, which comprises - a peptide of any one of claims 1 to 1 A pharmaceutical composition having an antioxidant activity, comprising a peptide according to any one of claims 1 to 10. 13. A pharmaceutical composition for inhibiting proliferation of cancer cells, which comprises a patent application scope The peptide of any one of items 1 to 10. 14. The pharmaceutical composition of claim 3, wherein the cancer cell is a gastric adenocarcinoma cell. 1 5. A pharmaceutical composition having anti-inflammatory activity, comprising A peptide according to any one of claims 1 to 10, wherein the food product comprises a peptide according to any one of the claims pp. 56 ❹ 201043239. A method for preparing a peptide having a right L + , an amino acid sequence as shown in SEQ ID NO: 1, which comprises: providing a spirulina; algae of genus algae material The algae material, the .m leaf, is hydrolyzed by a protease, thereby obtaining a hydrolyzate; and the ~hydrolyzate is subjected to a purification treatment, thereby obtaining the month too. The method of claim 17, wherein the chlorella algae is a group consisting of sputum j: chlorella ((5) (four) "" coffee, protein nucleus, chlorella (Chlorella) EIlips〇ide illusion, respect], small to thin 〇 〇 α α α 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖 咖19. The method of claim i, wherein the chlorella algae is Chlorella vulgaris. 20. The method of claim 17, wherein the algae material is a natural material. 21. The method of claim 17, wherein the algae material is subjected to a processing prior to being hydrolyzed. 22. The method of claim 21, wherein the processing is selected from the group consisting of water extraction treatment, enzyme hydrolysis treatment, heat treatment, high temperature and high pressure treatment, high pressure homogenization treatment, acid treatment, and treatment treatment. And freeze thawing treatment, and combinations thereof. 23. The method of claim 17, wherein the peptone is selected from the group consisting of 57 201043239: pepsin, basal peptone, Λ ϊ ϊ 丨 & small " melon acid _ ' A ! Flavor proteases, and combinations thereof. 24 = The method of claim 23, wherein the protein is a method of stomach protein 25.= Patent Application No. 17, wherein the purification treatment is selected from the group consisting of two columns: ammonium sulfate fractionation Treatment, gelation, ion exchange chromatography, read phase, P, „ ^ reverse phase chromatography, hydrophobic interaction chromatography M, and combinations thereof.
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