這篇論文是出自於美國FDA旗下 的一個接近醫學期刊論文級別的網站， 是比較可信的研究報告。
我的高中同學Dr.T 是日本生化博士，以下是他的po文。
紅茶與普洱茶，抗冠狀病毒！
嗜喝咖啡/烏龍茶/綠茶的朋友，最近可以改喝紅茶或是普洱。
植物總是在人不經意的時候，提供適時的幫助。紅茶跟普洱所含的茶黃素（TF3），不僅形成紅茶深邃的顏色，也能夠阻止冠狀病毒的複製。而烏龍與綠茶則沒有這個效果。
日本人真的很厲害，產官學合作開發的とろみ紅茶，加入纖維，提升2～3倍的TF3，原訴求抗流感病毒，現在用來抗冠狀病毒。另外還有含TF3的喉糖～～可以多吃點。
論文出處 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142193/#__ffn_sectitle
Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3′-digallate (TF3)
茶黃素-3,3′-對苯二甲酸酯（TF3）對SARS-CoV 3C樣蛋白酶活性的抑製作用
SARS-CoV是嚴重急性呼吸道綜合症（SARS）的病原體。病毒編碼的3C樣蛋白酶（3CLPro）被認為對SARS-CoV在受感染宿主細胞中的病毒複製至關重要。在這項研究中，我們篩選了由720種化合物組成的天然產物庫，該庫具有針對3CLPro的抑制活性。庫中發現有兩種化合物具有抑製作用：單寧酸（IC50 = 3 µM）和3-異theflaflavin-3-gallate（TF2B）（IC50 = 7 µM）。這兩種化合物屬於茶中發現的一組天然多酚。我們進一步研究了幾種不同類型茶（包括綠茶，烏龍茶，普er茶和紅茶）提取物的3CLPro抑制活性。我們的結果表明，普Pu茶和紅茶的提取物在抑制3CLPro方面比綠茶或烏龍茶更有效。還評估了茶中其他幾種已知的組合物抑制3CLPro的活性。我們發現咖啡因，（-）-表沒食子兒茶素沒食子酸酯（EGCg），表兒茶素（EC），茶鹼（TP），兒茶素（C），表兒茶素沒食子酸酯（ECg）和表沒食子兒茶素（EGC）不會抑制3CLPro活性。發現只有茶黃素-3,3′-對苯二甲酸酯（TF3）是3CLPro抑製劑。這項研究已導致鑑定出有效的3CLPro抑製劑新化合物。
SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS). The virally encoded 3C-like protease (3CLPro) has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM) and 3-isotheaflavin-3-gallate (TF2B) (IC50 = 7 µM). These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—)-epigallocatechin gallte (EGCg), epicatechin (EC), theophylline (TP), catechin (C), epicatechin gallate (ECg) and epigallocatechin (EGC) did not inhibit 3CLPro activity. Only theaflavin-3,3′-digallate (TF3) was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.
介紹：
嚴重急性呼吸系統綜合症（SARS）由新發現的冠狀病毒SARS冠狀病毒（SARS-CoV）引起（1,2）。從2002年11月1日到2003年6月18日，來自29個國家的世界衛生組織共報告了8465例可能的SARS病例，有801人死於該疾病。病死率約為10％。儘管已經避免了病毒的傳播，但SARS的重新出現仍存在疑問（3–6）。
像其他相關的冠狀病毒一樣，SARS-CoV是一種包膜病毒，其中包含正極性的單鏈RNA。冠狀病毒是RNA病毒中已知最大的基因組（約30 kb）[7]。 SARS-CoV的基因組包含一個開放閱讀框（ORF），該開放閱讀框編碼兩個重疊的複制酶多蛋白多蛋白-1a（pp1a，約450 kDa）和poplyprotein-1ab（pp1ab，約750 kDa），負責病毒複製（7）。基因組的其餘部分包括至少五個編碼結構蛋白和其他具有推定功能的基因的ORF（8,9）。蛋白水解過程，尤其是複制酶多蛋白的過程，是許多正鏈RNA病毒（包括冠狀病毒）生命週期中的關鍵步驟之一。所有冠狀病毒都編碼在病毒成熟期間用於蛋白水解過程的脯氨酸樣蛋白酶（PLP）和胰凝乳蛋白酶樣（3CLPro）蛋白酶（10）。對其他冠狀病毒的研究表明，PLP蛋白在pp1a多蛋白上不少於兩個位點裂解，而3CLPro蛋白酶在pp1a和pp1ab多蛋白上裂解至少11個域間位點[7]。因此，SARS-CoV的3CLPro蛋白酶已被認為是抗SARS-CoV藥物發現和開發的重要分子靶標。在這項研究中，通過篩選天然產物庫並進一步確認，我們發現SARS-3CLPro可以被茶中富含的化合物抑制。我們還檢查了各種茶和茶中一組代表性天然產品的粗提取物對SARS-3CLPro的抑制活性。
Severe acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus, SARS coronavirus (SARS-CoV) (1,2). From November 1, 2002 to June 18, 2003, a total of 8465 probable SARS cases were reported to the World Health Organization from 29 countries, and 801 people died from this disease. The case–fatality proportion was ∼10%. Although the spread of the virus has been averted, the re-emergence of SARS is still in question (3–6).
Like other related coronaviruses, SARS-CoV is an enveloped virus containing single-stranded RNA of positive polarity. Coronaviruses have the largest genomes known (∼30 kb) among RNA viruses [7]. The genome of SARS-CoV comprises one open reading frame (ORF) encoding two overlapping replicase polyproteins, polyprotein-1a (pp1a, ∼450 kDa) and poplyprotein-1ab (pp1ab, ∼750 kDa), responsible for virus replication (7). The rest of the genome comprises at least five ORFs encoding structural proteins and other genes with putative functions (8,9). Proteolytic processing, especially the processing of replicase polyproteins, is one of the crucial steps in the life cycle of many positive-stranded RNA viruses, including coronaviruses. All coronaviruses encode a papline-like protease (PLP) and a chymotrypsin-like (3CLPro) protease for proteolytic procession during virus maturation (10). Studies on other coronaviruses have shown that the PLP protein cleaves at no less than two sites on the pp1a polyprotein and that the 3CLPro protease cleaves at least 11 inter-domain sites on the pp1a and pp1ab polyproteins [7]. Thus, the 3CLPro protease of SARS-CoV has been considered as an important molecular target for anti-SARS-CoV drug discovery and developments. In this study, after screening of a natural product library and further confirmation, we found that SARS-3CLPro could be inhibited by compounds that are abundant in teas. We also examined crude extracts from various teas and a panel of representative natural products in teas for their inhibitory activities against SARS-3CLPro.
SARS-CoV 3CLPro的製備
從SARS患者的咽拭子中提取的總RNA由台灣台北市疾病控制中心（CDC）提供。所有RNA樣品均按照生物安全2級（BSL2）規定進行處理。兩種引物，即5′-GGCGGAATTCGCCTCTACCAACCACCACAGA-3’和5′-GGCGGAATTCA AAGCATCCAATGATGAGTGCC-3’被用於逆轉錄聚合酶鏈反應（RT-PCR），使用Ready-to-Go RT-方法合成SARS-3CLPro的cDNA。根據製造商的方案，PCR珠子（Amersham Biosciences，Piscataway，NJ）。用於擴增SARS-3CLPro cDNA的PCR循環如下：94°C 5分鐘，25個94°C 1分鐘，58°C 3分鐘，72°C 2分鐘和72°C的循環。 C持續10分鐘。 SARS冠狀病毒的全長3CLPro已作為穀胱甘肽S轉移酶（GST）融合蛋白在大腸桿菌中成功表達。凝血酶切割後，將3CLPro純化至均質。合成具有對應於SARS-3CLPro的自動切割位點的序列H2N-SITSAVLQSGFRKMA-COOH的15聚體肽，並將其用於蛋白酶切割活性測定。高效液相色譜（HPLC）分析顯示高度純化的蛋白質具有反式蛋白水解活性。
複合集合
用於篩選3CLPro抑製作用的Pure Natural Products庫是從MicroSource Discovery Systems，Inc.，Gaylordsville，CT獲得的。它包含720種純天然產品的獨特收藏。該館藏包括簡單和復雜的氧雜環，生物鹼，七萜烯，二萜，五環三萜，固醇和許多其他不同的代表。最初，收集了10種不同的化合物，並測試了混合物對SARS-3CLPro的抑制活性。然後對正庫進行反捲積，將能夠在10 µM抑制大於50％的肽裂解的單一純化合物記為命中。其他化合物獲自Sigma（St Louis，MO）。茶黃素（TF1），TF2 [TF-2A（theaflavin-3-gallate）和TF-2B（theaflavin-3′-gallate的混合物）]和theaflavin-3,3′-digallate（TF3）是Dr的好禮物梁玉芝，台北醫科大學醫學院，台灣，中華民國。
烏龍茶，綠茶，紅茶和普er茶粗提物的製備
茶液由烏龍茶，綠茶，紅茶和普er茶（也稱為普-茶或普-茶）樣品製成，含量為2％（w / v）。通過在熱燒瓶中在沸騰的熱水中搖動10分鐘來製備四種不同類型的茶葉的水提取物。然後將提取物通過Millex-GS 0.22 µm過濾器（Millipore，法國馬爾斯海姆）過濾，以除去顆粒物。然後在減壓下蒸發水提取物以獲得粘性物質。評價這些材料對SARS-3CLPro的抑制活性。
HPLC法抑制SARS-3CLPro蛋白酶活性
通用半胱氨酸蛋白酶抑製劑N-乙基馬來酰亞胺（NEM，蛋白酶抑製劑作為陽性對照）獲自Sigma（密蘇里州聖路易斯）。在肽裂解試驗中測試了該化合物對SARS-3CLPro的抑制活性。在不存在或存在不同濃度的這些通用抑製劑的情況下測量蛋白酶的活性。將酶（2 µM）與化學物質預孵育30分鐘。然後將100 µM的肽加入到反應中，並將混合物在37°C孵育1小時。計算了抑製劑的效力。 HPLC分析條件為：C18 RP保護柱（250×4.6 mm×5 µm，Agilent Zorbax Extend）。選擇此方法是因為它在幾種測試方法中提供了最佳分辨率。柱溫為環境溫度，使用包含溶劑A [10 MM NH4OAc，0.1％三氟乙酸（TFA）]和B（乙腈，0.1％TFA）混合的溶劑系統進行洗脫（0.8 ml / min）從100％A開始，在8分鐘內線性下降到10％A，在16分鐘內線性下降到100％A，並在100％A下保持23分鐘。注射體積為40ml。紫外線檢測器設置在214 nm。
熒光底物肽檢測SARS-3CLPro的蛋白水解活性
先前已經報導了熒光蛋白酶活性測定法的發展（7）。使用熒光酶標儀（Fluoroskan Ascent，來自ThermoLabsystems，Sweden），在538nm處激發並在538nm下監測因蛋白酶催化的熒光底物肽（Dabcyl-KTSAVLQSGFRKME-Edans）的裂解而引起的增強熒光。熒光測定法用於確定已鑑定的抑製劑對SARS 3CLPro活性的IC50。蛋白酶在使用前儲存在-70°C的緩衝液中，該緩衝液包含12 mM Tris-HCl（pH 7.5），120 mM NaCl，0.1 mM EDTA，7.5 mM 2-巰基乙醇和1 mM二硫蘇糖醇（DTT）。
Preparation of SARS-CoV 3CLPro
Total RNA extracted from a throat swab of a SARS patient was provided by the Center for Disease Control (CDC), Taipei, Taiwan. All RNA samples were handled under bio-safety level 2 (BSL2) regulations. Two primers, 5′-GGCGGAATTCGCCTCTACCAACCACCACAGA-3′ and 5′-GGCGGAATTCA AAGCATCCAATGATGAGTGCC-3′, were used for reverse transcription–polymerase chain reaction (RT–PCR) to synthesize the cDNA of SARS-3CLPro using Ready-to-Go RT–PCR Beads (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s protocol. The PCR cycle used for the amplification of cDNA of SARS-3CLPro was as follows: 94°C for 5 min, 25 cycles of 94°C for 1 min, 58°C for 3 min, 72°C for 2 min and 72°C for 10 min. A full-length 3CLPro of SARS coronavirus was successfully expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Following cleavage by thrombin, 3CLPro was purified to homogeneity. A 15mer peptide with sequence H2N-SITSAVLQSGFRKMA-COOH corresponding to an autocleavage site of the SARS-3CLPro was synthesized and used for protease cleavage activity assay. The highly purified protein was shown by high-performance liquid chromatography (HPLC) analysis to exhibit proteolytic activity in trans.
Compound Collection
The Pure Natural Products Library used to screen for 3CLPro inhibition was obtained from MicroSource Discovery Systems, Inc., Gaylordsville, CT. It consists of a unique collection of 720 pure natural products. The collection includes simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentacyclic triterpenes, sterols and many other diverse representatives. Initially, 10 different compounds were pooled, and the mixtures were tested for their inhibitory activities against SARS-3CLPro. Positive pools were then deconvoluted and single pure compounds that were able to inhibit >50% of peptide cleavage at 10 µM were scored as hits. Other compounds were obtained from Sigma (St Louis, MO). Theaflavin (TF1), TF2 [a mixture of TF-2A (theaflavin-3-gallate) and TF-2B (theaflavin-3′-gallate)] and theaflavin-3,3′-digallate (TF3) were kind gifts from Dr Yu-Chih Liang, School of Medical Technology, Taipei Medical University, Taipei, Taiwan, ROC.
Preparation of Crude Extracts from Oolong Tea, Green Tea, Black Tea and Puer Tea
Tea liquors were prepared from oolong tea, green tea, black tea and Puer tea (also called pu-er or pu-erh) samples as 2% (w/v) tea solutions. The water extracts of four different types of tea leaves were prepared by shaking for 10 min in boiling hot water in thermal flasks. The extracts were then filtered through a Millex-GS 0.22 µm filter (Millipore, Malsheim, France) to remove particulate matter. The water extracts were then evaporated under reduced pressure to obtain viscous masses. These materials were evaluated for their inhibitory activities against SARS-3CLPro.
Inhibition of Protease Activity of SARS-3CLPro by HPLC Assay
A general cysteine protease inhibitor, N-ethylmaleimide (NEM, protease inhibitor as positive control), was obtained from Sigma (St Louis, MO). This compound was tested for its inhibitory activities against SARS-3CLPro in the peptide cleavage assay. The activities of the proteases were measured in the absence or presence of different concentrations of these general inhibitors. The enzyme (2 µM) was pre-incubated with chemicals for 30 min. Peptide was then added at 100 µM to the reaction, and mixtures were incubated at 37°C for 1 h. The potency of the inhibitors was calculated. The HPLC analysis condition is: a C18 RP guard column (250 × 4.6 mm × 5 µm, Agilent Zorbax Extend). This method was chosen because it gave the best resolution among several methods that were tested. The column temperature was ambient, and the elution (0.8 ml/min) was performed using a solvent system comprising solvents A [10 MM NH4OAc, 0.1% trifluoroacetic acid (TFA)] and B (acetonitrile, 0.1% TFA) mixed using a gradient starting with 100% A, linearly decreasing to 10% A in 8 min, to 100% A in 16 min, and held at 100% A for 23 min. The injection volume was 40 ml. The UV detector was set at 214 nm.
Proteolytic Activity of SARS-3CLPro by Fluorogenic Substrate Peptide Assay
The development of a fluorescent protease activity assay has been reported previously (7). The enhanced fluorescence due to cleavage of the fluorogenic substrate peptide (Dabcyl-KTSAVLQSGFRKME-Edans) catalyzed by the protease was monitored at 538 nm with excitation at 355 nm using a fluorescence plate reader (Fluoroskan Ascent from ThermoLabsystems, Sweden). The fluorimetric assay was used to determine the IC50 of identified inhibitors on SARS 3CLPro activity. The protease was stored in the buffer containing 12 mM Tris–HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA, 7.5 mM 2-mercaptoethanol and 1 mM dithiothreitol (DTT) at −70°C before use.
茶提取物和茶中純成分抑制3CLPro活性的評價
由於各種茶中還存在許多與鞣酸和TF2B有關的其他多酚，因此我們決定研究茶中存在的各種茶提取物和幾種眾所周知的純淨成分對活性的抑製作用。因此，製備了來自不同類型茶的水提取物，並評價了其對3CLPro的抑制活性。如表1所示，來自紅茶和普er茶的提取物抑制3CLPro活性，而來自綠茶或烏龍茶的提取物則沒有。我們進一步測試了茶中存在的幾種已知成分可能參與其中，包括咖啡因，茶鹼，兒茶素（C），表沒食子兒茶素（EGC），（-）-表沒食子兒茶素沒食子酸酯（EGCg），表兒茶素（EC），表兒茶素沒食子酸酯（ECg）， TF1，TF2，TF2B，鞣酸和TF3。如表1所示，結果表明甲基黃嘌呤（咖啡因和茶鹼）不影響3CLPro的活性。綠茶（未發酵）和烏龍茶（部分發酵）中存在的兒茶素（包括C，EC，ECg，EGC和EGCg）也不會抑制3CLPro活性。我們的結果表明單寧酸，TF2B和TF3是3CLPro抑製劑，如熒光底物測定所揭示（圖3）。 TF2A由於不可用而未經測試。
Evaluation of Tea Extracts and Pure Components from Teas for Their Inhibition of 3CLPro Activity
Since many other polyphenols related to tannic acid and TF2B are also present in various kinds of teas, we decided to examine the inhibition of activity by various tea extracts and several well known pure ingredients present in teas. Thus, water extracts from different types of teas were prepared and evaluated for their inhibitory activities against 3CLPro. As shown in Table 1, extracts from black and Puer teas inhibited 3CLPro activity while those from green or oolong teas did not. We further tested the possible involvement of several known ingredients present in teas, including caffeine, theophylline, catechin (C), epigallocatechin (EGC), (—)-epigallocatechin gallte (EGCg), epicatechin (EC), epicatechin gallate (ECg), TF1, TF2, TF2B, tannic acid and TF3. As shown in Table 1, the results showed that methylxanthine (caffeine and theophylline) did not influence 3CLPro activity; catechins, including C, EC, ECg, EGC and EGCg, present in green tea (unfermented) and oolong tea (partial fermented) also did not inhibit 3CLPro activity. Our results indicated that tannic acid, TF2B and TF3 are 3CLPro inhibitors as revealed by the fluorogenic substrate assay (Fig. 3). TF2A was not tested because of its unavailability.
TF2B和TF3作為抗3CLPro抑制活性的有效劑
天然產物篩選的文庫由簡單和復雜的氧雜環，生物鹼，半萜烯，二萜，五環三萜，甾醇和許多其他不同的代表組成。發現許多紅茶多酚（尤其是TF1，TF2和TF3的多酚）抑制3CLPro。在紅茶中，TF3含量最高（1.05％），其次是TF2A（0.34％），TF2B（0.11％）和TF1（0.08％）（16）。有趣的是，如圖3b所示，TF3包含連接在3,3’位置的兩個沒食子酸酯基團。另一方面，TF2A和TF2B僅由一個分別連接到3或3’位置的沒食子酸酯基組成（圖4b）。相反，沒有沒食子酸酯基團附著於TF1。表1中的結果表明，連接至TF2B和TF3的3’位的沒食子酸酯基對於與3CLPro活性位點相互作用可能很重要。 EC，EGCg，ECG和EGC的活性（茶黃素50≥100 µM）遠低於茶黃素。
TF2B and TF3 as Potent Agents Against 3CLPro Inhibitory Activity
The natural product screened library consisted of simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentacyclic triterpenes, sterols and many other diverse representatives. Numerous black tea polyphenols (especially those of TF1, TF2 and TF3) were found to inhibit 3CLPro. In black tea, TF3 was the most abundant (1.05%) followed by TF2A (0.34%), TF2B (0.11%) and TF1 (0.08%) (16). It is interesting to note that TF3 contained two gallate groups attached to the 3,3′ positions as shown in Fig. 4b. TF2A and TF2B, on the other hand, consisted of only one gallate group attached either to the 3 or the 3′ positions, respectively (Fig. 4b). In contrast, no gallate group was attached to TF1. Results in Table 1, indicating that the gallate group attached to the 3′ position to TF2B and TF3, might be important for interaction with the 3CLPro active site. EC, EGCg, ECG and EGC were much less active (IC50 ≥ 100 µM) than theaflavins.
討論區
人冠狀病毒（HCoV）是包括人在內的動物上呼吸道疾病的主要原因。已證明新型冠狀病毒可引起SARS。主要蛋白酶（通常稱為3C樣蛋白酶（3CLPro））已被確定為有吸引力的藥物靶標（17）。在這項研究中篩選的720種天然產物中，使用HPLC蛋白水解測定法鑑定了鞣酸（圖4a）和TF2B，以抑制濃度≤10 µM的3CLPro的50％的蛋白水解活性。單寧酸和TF2B均屬於茶中的天然多酚類。發現低濃度的鞣酸可抑制蛋白酶，包括組織型纖溶酶原激活物，尿激酶型纖溶酶原激活物和纖溶酶活性[13]。現在生產了300多種茶，但將它們分為三種主要形式：綠茶是通過乾燥新鮮的茶葉製成的，重點是在製造過程中防止茶多酚的氧化。相反，紅茶的生產以高度發酵為特徵，其在茶中存在的成分之間產生一系列化學縮合。烏龍茶經過部分發酵，其茶成分與綠茶中的成分更為相似（18）。在評估不同類型的茶時，我們發現幾種不同類型的茶（包括普er茶和紅茶）的提取物可以抑制3CLPro活性，而綠茶或烏龍茶則不能。然而，普er茶中的化學成分非常複雜，很難分離出純淨的成分進行結構鑑定。普er茶成分的純化和鑑定應在單獨的研究中進行。
接下來，我們檢查了茶中存在的其他知名成分是否也可以抑制3CLPro活性。我們發現甲基黃嘌呤（咖啡因和茶鹼）和兒茶素（EGCg，EC，C，ECg和EGC）在濃度高達100 µM時不能抑制3CLPro的活性（表1）。但是，TF1，TF2和TF3是比綠茶中的兒茶素更有效的3CLPro抑製劑。在生產紅茶的發酵步驟中，大多數兒茶素被氧化並通過二聚作用縮合到茶黃素中，並通過聚合反應縮合到茶紅素中。綠茶中含有約30％的兒茶素（乾基），而紅茶中含有約9％的兒茶素和4％的茶黃素（19）。在另一項研究中，Leung等人。 （16）報告說，TF3是紅茶中茶黃素含量最高的（1.05％），其次是TF2A（0.34％），TF2B（0.11％）和TF1（0.08％）。單寧酸是植物中的一類多酚，但是由於單寧酸構成了多種含摩爾的聚合物，因此很難對綠茶或紅茶中其含量進行定量。 wt– 500–3000 Da。
有趣的是，與TF1相比，TF2B和TF3是3CLPro的更有效抑製劑（表1）。與TF2B和TF3不同，TF1不包含沒食子酸酯基團（圖4b）。因此，在TF2B和TF3的39位上連接一個沒食子酸酯基可能對它們對3CLPro的抑制活性很重要。這些結果表明，TF2B和TF3可能是設計SARS-CoV編碼的3CLPro活性更高的抑製劑的良好起點。
最後，這項研究確定了有效的3CLPro抑製劑（IC50≤10µM）的三種化合物（TF2B，TF3和單寧酸）。這些化合物在紅茶提取物中含量豐富（16，19）。紅茶是世界上流行的飲料。這項研究的結果值得進一步研究，以檢查這些天然產物在細胞培養中抑制SARS-CoV複製的作用。克拉克等。報導稱，從紅茶中提取的茶黃素能夠中和牛冠狀病毒和輪狀病毒感染（20）。因此，在另一項研究中評估飲用紅茶是否可以預防或減輕腸道形式的冠狀病毒感染將是非常有趣的，因為已知SARS-CoV可以在腸道中主動複製。
Discussion
Human coronaviruses (HCoVs) are major causes of upper respiratory tract illness in animals including humans. It has been demonstrated that a novel coronavirus causes SARS. The main protease, frequently called 3C-like protease (3CLPro), has been identified as an attractive drug target (17). Of the 720 natural products screened in this study, tannic acid (Fig. 4a) and TF2B were identified using the HPLC proteolytic assay to inhibit 50% of the proteolytic acitivity of 3CLPro at concentrations ≤10 µM. Both tannic acid and TF2B belong to the group of natural polyphenols found in teas. Low concentrations of tannic acid were found to inhibit proteases including tissue-type plasminogen activator, urokinase-type plasminogen activator and plasmin activity [13]. Over 300 different kinds of tea are now produced, but they are classified into three main forms: green tea is manufactured by drying fresh tea leaves, with emphasis on the prevention of oxidation of the tea polyphenols during the manufacturing process. In contrast, the manufacture of black tea is characterized by a high degree of fermentation, which produces a series of chemical condensations among the ingredients present in tea. Oolong tea is partially fermented, and the tea compositions are more similar to those found in green tea (18). In evaluating different types of tea, we found that extracts from several different types of tea, including Puer and black tea, could inhibit 3CLPro activity while green or oolong tea could not. However, the chemical composition in Puer tea is very complex and it is difficult to isolate pure ingredients for structural identification. Purification and identification of Puer tea ingredients should be carried out in a separate study.
We next examined whether other well-known ingredients present in tea could also inhibit 3CLPro activity. We found that methylxanthine (caffeine and theophylline) and catechins (EGCg, EC, C, ECg and EGC) were not able to inhibit 3CLPro activity at concentrations up to 100 µM (Table 1). However, TF1, TF2 and TF3 were more potent 3CLPro inhibitors than catechins in green tea. During the fermentation step in the production of black tea, most of the catechins are oxidized and condensed into theaflavins through dimerization and into thearubigins through polymerization. Green tea contains ∼30% of catechins (dry mass base) while black tea contains ∼9% of catechins and 4% of theaflavins (19). In another study, Leung et al. (16) reported that TF3 was the most abundant (1.05%) theaflavin in black tea followed by TF2A (0.34%), TF2B (0.11%) and TF1 (0.08%). Tannic acid is a class of polyphenolic in plants but the quantification of its levels in green or black tea is difficult because tannic acid constitutes a wide range of polymers with mol. wts of 500–3000 Da.
It is interesting to note that TF2B and TF3 are more potent inhibitors of 3CLPro than TF1 (Table 1). Unlike TF2B and TF3, TF1 does not contain a gallate group (Fig. 4b). Thus, the addition of a gallate group attached at the 39 position to TF2B and TF3 might be important for their inhibitory activity against 3CLPro. These results suggest that TF2B and TF3 might be good starting points for the design of more active inhibitors for the 3CLPro encoded by SARS-CoV.
Finally, this study has identified three compounds (TF2B, TF3 and tannic acid) that are effective 3CLPro inhibitors (IC50 ≤10 µM). These compounds are abundant in the extract of black tea (16,19). Black tea is a popular beverage in the world. Results from this study warrant further investigation to examine the effect of these natural products in inhibition of SARS-CoV replication in cell culture. Clark et al. reported that theaflavins extracted from black tea were able to neutralize bovine coronavirus and rotavirus infections (20). Thus, it will be very interesting to evaluate, in a separate study, whether drinking black tea can prevent or alleviate the infection of an enteric form of coronavirus since SARS-CoV is known to actively replicate in the intestinal tract
參考閱讀：https://blog.xuite.net/boavista/blog/588859763 ►大陸最新發現 雙黃連口服液可抑制武漢肺炎