Polyphenol content and serine-Protease inhibitory activities from selected medicinal plants growing in Vietnam - Nguyen Minh Thang

TAP CHI SINH HOC 2015, 37(1se): 1-11 DOI: 10.15625/0866-7160/v37n1se. POLYPHENOL CONTENT AND SERINE-PROTEASE INHIBITORY ACTIVITIES FROM SELECTED MEDICINAL PLANTS GROWING IN VIETNAM Nguyen Minh Thang, Pham Thi Tran Chau* Vietnam National University, Hanoi, *phamthitranchau@gmail.com ABSTRACT: Proteinase inhibitors play a crucial role in the regulation of metabolism and the development of living beings. This research deals with proteinase inhibitory activity (PIA) and polyphenol contents from 20 Vietnamese medicinal plants, which have been used to cure infection, pimples and rash. The relationship between PIA and polyphenol content and their bioavailability were investigated. Among the 26 aqueous fresh samples of the 20 medicinal plants that belong to 17 different families, we found 21 samples from 16 plants possess inhibitory activity against trypsin (TIA), chymotrypsin (ChIA) and extracellular proteases from Pseudomonas aeruginosa (PsIA), of which ChIA is found to be highest in this respect. In the same connection, Syzygium resinosum Gagnep has the highest PIA (TIA, PsIA, ChIA about 11631, 8195, 33332 mIU/g fresh sample, respectively). There have been found that the samples with high PIA also possess high content of polyphenol. PIA of ethanol extracts is higher than that of aqueous extracts. An investigation of inhibitory activity against PA of P. aeruginosa and Staphylococcus aureus by zymography has indicated that ethanol extracts of sappan heartwood and dormant seed coat can put an end to or partially reduce all three proteinase bands from these microbes. For further study, heartwood flavonoid is fractionated on silicagel column chromatography. All obtained fractions exhibit PIA, and the major fraction is taken for subsequent purification and then subjected to a NMR analysis. The result of the analysis shows that the fraction appears to be protosappanin A. Keyword: Pseudomonas aeruginosa, Staphylococcus aureus, Caesalpinia sappan, protease, protease inhibitor, polyphenol, flavonoid, infection disease. INTRODUCTION Proteolytic enzymes play a crucial role in living systems. So far, polypeptides that have proteolytic activity reach about 140,000 chains [17]. Many diseases such as infections, heart diseases, cystic fibrosis, cancers are related with protease or malfunction of protease. It is reported that there are 80 genetic diseases in human caused by mutation of protease genes, metastasis of tumor has also resulted from proteinase malfunction [19]. The proteolytic enzymes are also actively participating in the process of inflammation in human beings. The high reaction of inflammation may cause tissues damage, necrosis [1, 20, 21]. Protein degradation ability of protease is magnificently high, thus nature adapts many level of regulations: gene regulation, secretion, activation, or inhibition. Plants possess efficient mechanisms against pathogen and pathogenic proteases enzyme system. These mechanisms have included the secondary metabolism with polyphenol compounds and their derivatives. Polyphenol is well-known as the antioxidant agent, and research has shown that polyphenol derivatives protect protein and genetic materials against free radical [3]. Polyphenol, specifically the flavonoid may interact to protein/enzyme in different ways which reduce cell damage, tumor formation and metastasis [18]. The high molecular polyphenol so call tannin can be unspecifically attached to protease resulting in precipitation and denaturation of protein [2, 20]. Other compounds can mimic the substrate then act as a specific inhibitor, these inhibitors show high potential for pharmaceutical application with a lower side effects than the synthesis compounds. Oriental medicine also mainly relies on the bioactivity of plant secondary compounds. The combination of plant compounds leads to the marvelous effect to many diseases that outweigh the Western medicine. In Vietnam, many plants have been used for treatment of microorganism infection (pimples, rash, itchiness) for some hundreds years already . Pathogenic microorganisms rely on their extracellular enzymes, particularly in the case of proteases to get a nutrition intake, therefore we hypothesize that the protease inhibitors that are innately existing in plants, inhibit microorganism development by way of reducing the sources of nutrition that are necessary for them. With a view to investigating the relationship between polyphenol and proteinase inhibitory activity from medicinal plants that cure infection, pimples and rash [24], in this work we focus on a study of polyphenol content and serine proteinases inhibitory activities from medicinal plants growing in Vietnam, particularly a study on sappanwood (Caesalpinia sappan L.). MATERIALS AND METHODS Material The samples collected from parts of Vietnam tradition medicinal plants provided by the Center of Cultivation and Processing of Medicinal Plants, Vietnam National Institute of Medicinal Materials. Instruments and chemicals Germany rotary evaporator Ika; Silicagel Thin layer plate DC alufolien 20x20 cm silicagel 60 F254 from Merck, Hoefer SE 260 electrophoretic system; HeλIOS a Spectronic Unicam spectrometer (England); DT thermal stabilizer (Denmark). Chemicals reach the PA grade. Methods Sample preparation for testing PIA and polyphenol content Fresh samples: Medicinal plants are collected, cleaned and cut into small pieces before being extracted with distilled water. Dried samples: Samples are treated at 100°C in 10 minutes for denaturing enzymes and dried at 60°C. The dried samples have extracted 3 times with 96% ethanol at room temperature for a week. The whole extracts are collected and evaporated using rotary evaporator to reach certain volume, and they are named total polyphenol extracts. Total flavonoid is isolated according to B. C. Talli method as described previously [12]. Determination of polyphenol content using Folin-Ciocalteau method [26]. From each calibration solution, sample, or blank, pipet 20 μL into separate cuvettes, 1.58 mL of water are added, then 100 μL of the Folin-Ciocalteau reagent is put into each cuvette and mix well. Wait for 30 seconds, and then add 300 μL of the 20% sodium carbonate solution, mix well. Leave the solutions at 40°C for 30 min and measure the absorbance of each solution at 765 nm against the blank. Polyphenol content is estimated by using gallic standard curve. Separation of polyphenol/flavonoid on thin layer chromatography (TLC) Determination of proteinase inhibitory activity (PIA) Diffusion method (for preliminary investigation) [9]. Modified Anson method [16]; One inhibitory unit (IU) is the amount of inhibitor which reduces the activity of two mg of trypsin by 50%. Proteolytic activity electrophoresis on polyacrylamide gel using Heussen and Dowdle method [4]. Silicagel column chromatography Silicagel beads with a size of 0.040-0.063 mm were used as a solid phase, solvent system is chloroform:ethyacetate:formic (5:3:0.4) and chloroform : ethylacetate (5:3). The collected fractions were tested on thin layer chromatography (TLC) with a solvent system: chloroform:ethyacetate:formic (5:3:0.4) Ultraviolet absorbance spectroscopy Each group of flavonoid possesses a typical absorbance spectrum. Chromatography fractions are diluted in ethanol and taken to UV-Vis analysis. NMR spectroscopy NMR spectroscopy is performed in a cooperation with Laboratory of Structure Chemistry, Institute of Chemistry, Vietnam Academy of Science and Technology RESULTS AND DISCUSSION Total polyphenol content and proteinase inhibitory activity (PIA) in selected medicinal plants Preliminary investigation of PIA To preliminarily study PIA from samples, the diffusion method has been used because of its rapid and sensitive characteristic. The results are shown in table 1, the samples which possess high PIA (against 3 studied serine proteinases: trypsin, chymotrypsin and extracellular proteinase(s) from Pseudomonas aeruginosa (PsPA) are chosen for further study. Table 1. Preliminary investigation of PIA (using Diffusion method) from medicinal plants No. Plants Samples TIA ChIA PsIA 1 Zanthoxylum avicennae (Lamk.) (Rutaceae) Radix +++ +++ ++ 2 Chenopodium ambrosioides L. (Chenopodiaceae) Foliar - - - 3 Asarum balansae Franch. (Aristolochiaceae) Foliar - - - 4 Combretum quadrangulare Kurz. (Combretaceae) Foliar +++++ +++++ +++++ 5 Amorphophallus rivieri Dur. (Araceae) Foliar - - - 6 Mirabilis jalapa L. (Nyctaginaceae) Foliar - - - 7 Melia azedarach L. (Meliaceae) Bark ++ +++ ++ 8 Ouisqualis indica L. (Combretaceae) Bark +++ ++ + 9 Bischofia javanica Blume (Euphorbiaceae) Bark ++ ++ - 10 Mercuriadis leiocarpa Sieb et Zuee. (Euphorbiaceae) Foliar + + + 11 Syzygium resinosum Gagnep. (Myrtaceae) Foliar +++++ +++++ +++++ Branch +++ +++ +++ 12 Syzygium formosum var. ternifolium (Wall) Matsam (Myrtaceae) Foliar ++++ +++ ++++ 13 Rheum sp. (Polygonaceae) Tuber ++++ ++++ ++++ 14 Cajanus indicus Spreng. (Fabaceae) Fruitcover - - - Foliar +++ +++ ++ 15 Mangifera indica L. (Anacardiaceae) Bark +++ +++ ++ 16 Caesalpinia sappan L. (Fabaceae) Wood ++++ +++++ +++ Bark ++++ +++++ +++++ Green seed coat +++ +++ ++ Dormant seed coat +++++ +++++ +++++ 17 Tamarindus indica L. (Fabaceae) Bark + + - 18 Plumeria rubra L. (Apocynaceae) Foliar +++ - - 19 Prunella vulgaris L. (Lamiaceae) Foliar ++ +++ ++ Flower ++ +++ +++ 20 Artium lappa L. (Asteraceae) Radix + ++ + -: No inhibiton; +: inhibit 75%; +++++: inhibit 100% of proteolytic activity of 2.5 microgram of protease (trypsin equivalency). Table 2. Qualitative determination of Protease inhibitory activities of aqueous extracts from samples with high PIA No. Samples Polyphenol (mg/g fresh sample) TIA ChIA PsIA mIU/g fresh sample mIU/mg polyphenol mIU/g fresh sample mIU/mg polyphenol mIU/g fresh sample mIU/mg polyphenol 1 C. quadrangulare 20.1 7651 380.6 11960 5950 1789 89.0 2 M. azedarach 3.3 1375 416.7 2763 837.3 260 78.8 3 O. indica 5.5 263 47.8 1154 209.8 184 33.5 4 M. indica 5.7 222 38.9 171 30.0 179 31.4 5 S. resinosum 12.3 11631 945.6 33332 2709.9 8195 666.3 6 S. formosum var. ternifolium  8.2 2465 300.6 3694 450.5 1518 185.1 7 Rheum sp. 8.7 759 87.2 2021 23.2 274 11.8 8 C. indicus 3.9 632 162.0 805 206.4 372 95.4 9 M. indica 1.9 287 151.1 1033 543.7 63 33.2 10 C. sappan wood 5.5 14 2.6 29 5.3 22 4.0 11 C. sappan bark 7.5 7 0.9 19 2.5 13 1.7 12 C. sappan dormant seeds coat (SDSC) 2.1 2861 1362.4 11.454 5454.3 5183 2468.1 13 C. sappan Green seeds coat (SGSC) 1.4 255 182.1 3463 2473.6 1271 907.9 Among 26 samples from 20 medicinal plants that belong to 17 families have been used to treat infection, pimples and rash, 21 samples from 16 plants are found to possess PIA, accounting for 80% investigated plants. This rate leads to the suggestion of a contribution of protease inhibitors within plants to the pharmacology properties of plants against infection diseases. High PIA samples are chosen for further analysis by the modified Anson method (table 2). Polyphenol content was determined by the Folin-Ciocalteau method as described above. All the values shown in this article are calculated from three independent experiments (data not shown). Table 2 shows that, ChIA of almost all the studied samples is higher than TIA; PsIA is lowest. This may be due to P. aeruginosa proteinase sample obtained from supernatant of P. aeruginosa culture broth that contains several proteases including the proteases not being inhibited by the inhibitors in the sample extracts. Literature also indicates that P. aeruginosa produces a number of extracellular proteases [15]. Moreover, it can be noted that the samples with high PIA also have high content of polyphenol such as Combretum quadrangulare and Syzygium resinosum. However, the sappan dormant seed coat (SDSC) although contains very low amount of polyphenol but its PIA is highest among the four studied sappan samples. Moreover, the sappan samples express different protease inhibitory effect [5, 12], therefore it deserves further analysis. Total polyphenol and flavonoid content in ethanol extracts. Ethanol is used because of its ability of thorough extraction of almost all polyphenol compounds from samples. Moreover, high concentration of ethanol will precipitate unwanted high molecular weight compounds as proteinaceous protease inhibitors. Table 3 shows that polyphenol content in ethanol extracts (total polyphenol extract) is significantly higher than that of aqueous extracts. C. sappan bark possesses the highest of polyphenol content (96.84mg/g dried sample). Table 3. Total polyphenol and flavonoid contents in selected samples No. Samples Content (mg/g dried sample) Content (mg/g fresh sample) Polyphenol Flavonoid Polyphenol Flavonoid 1 S. resinosum 46.13 6.52 15.91 2.25 2 S. formosum var. ternifolium  62.41 16.11 21.51 5.55 3 Rheum sp. 53.57 20.21 5.58 5.58 4 C. indicus 13.43 1.78 3.71 0.49 5 M. indica 14.35 5.18 4.98 1.80 6 C. sappan wood 59.00 44.49 33.75 25.45 7 C. sappan bark 96.84 25.27 46.25 1207 8 SDSC 51.93 1.55 41.08 1.23 9 SGSC 21.39 1.99 7.19 0.67 Figure 1. Comparison of polyphenol content in aqueous and ethanol extracts of selected samples Figure 1 shows that in all studied cases, the total polyphenol content in ethanol extract is always higher than that in aqueous solution. Moreover, flavonoid content is rather low, in the range of 3% (SDSC) to 40% (Rheum sp.) of total polyphenol, the highest proportion belongs to C. sappan wood (75%). Table 4. Protease inhibitory activities of total polyphenol No. Samples TIA ChIA PsIA (mIU/g dried sample) mIU/mg polyphenol (mIU/g dried sample) mIU/mg polyphenol (mIU/g dried sample) mIU/mg polyphenol 1 S. resinosum 25553 554 73544 1594 28923 627 2 S. formosum var. ternifolium 12528 201 37707 604 10053 161 3 Rheum sp. 416 8 1388 26 454 9 4 C. indicus 4056 302 11957 890 4615 344 5 M. indica leiocarpa 3763 262 11549 804 2099 146 6 C. sappan wood 1976 34 16729 283 4925 84 7 C. sappan bark 13264 137 44311 458 1732 179 8 SDSC 74016 1425 530681 10217 99025 1906 9 SGSC 18733 890 126556 6015 25416 1208 Proteinase inhibitory activities of total polyphenol and flavonoid The total polyphenol from all samples inhibits all 3 studied proteinases, and SDSC sample shows highest inhibitory activity. PIA per 1 mg polyphenol from ethanol extracts is also higher than that of aqueous extracts with an exception of Rheum sp. and Syzygium formosum var. ternifolium (Wall) Matsam (table 4). PIA of flavonoid fraction from all samples extracts is much lower than that of the total polyphenol extracts (except Rheum sp.). Our previous study using SDS-PAGE and zymography has indicated that at least 7 protein protease inhibitory bands have been detected from C. sappan coat-free dormant seeds extracts [5]; however, no band has been found from C. sappan seeds coat extracts although protease inhibitory activity (PIA) of the latter is higher than that of the former. Thus, it means that PIA of seeds coat extract is not caused by its proteinaceous inhibitors but rather by other compounds with smaller molecular mass. In other article [12], we confirm that the inhibitory activity against protease is due to the act of several polyphenol compounds in parts of sappan. Moreover, specific inhibitory activity of polyphenol (being defined as mIU/mg polyphenol) from sappan seed coat is rather high. In the following part of this work, research is focused on a study of protease inhibitory activity of polyphenol compounds from C. sappan. Figure 2. Chromatogram of flavonoid extracts on TLC 1. C. sappan wood; 2. C. sappan bark; 3. SDSC; 4. SGSC; 5. S. resinosum Gagnep; 6. S. formosum var. ternifolium (Wall) Matsam; 7. Rheum sp.; 8. C. indicus Spreng; 9. M. indica L. Thin layer chromatography of Polyphenol/flavonoid from selected plants Flavonoid extracts are applied on silicagel thin layer plate and developed in different systems of solvent: Chloroform: Methanol (9:1); Toluence: Ethylacetate: Acetone: Formic (TEAF) (5:2:1:1) and TEAF(5:2:2:1). The result shows that the best resolution belonged to the TEAF (5:2:2:1) system. After chromatography, the plates are taken to dye with H2SO4 10% at 110°C in 10 minutes (figure 2). Bands are separately cut and extracted by using ethanol, then subjected to PIA test by using diffusion method. Experiment shows that PIA is concentrated at the bands with slow mobility, particularly at such places where the samples are loaded (detail for sappan samples were reported previously), and that the PIA compounds are more water dissolvable and/or have high molecular weight. Tannin content from C. sappan samples Tannin is a well-known compound in plant which has been used to tan animal skin because of its ability to make the cross link of the skin’s collagen. Tannin compounds have several types, of which molecular mass may reach 20,000 Da. The water soluble tannin compounds have molecular weight between 300 Da and 3,000 Da [8]. Tannin compounds are generally recognized as antimicrobial and non-specific protein binding compounds. Table 5. Tannins content in C. sappan samples Samples Tannins (mg/g dried sample) % Tannins in total polvphenol C. sappan wood 9.0 15.5 C. sappan bark 19.3 19.9 SDSC 13.8 26.6 SGSC 2.5 11.8 The above experiments bring about the hypothesis that tannin compounds may play a role in inhibitory activity of samples. Thus, the content of tannin compounds from C. sappan samples is investigated by the precipitation method using gelatin with a modification published recently in 2009 [13]. The content of tannin compounds from bark extracts is the highest, contributing 19.9% to its total polyphenol. However, SDSC tannin contributes 26.6% to its total polyphenol. SDSC possesses the lowest percentage of flavonoid but highest proportion of tannin and PIA, so tannin might be the major inhibitor from SDSC extracts (table 5). Proteases from P. aeruginosa and S. aureus As is known, P. aeruginosa and S. aureus produce several extracellular proteases involving in their pathogenesis [6, 22]. Novel protease from P. aeruginosa are also reported periodically [15, 7, 27]. Proteases from bacteria have been obtained by precipitating with acetone at low temperature, and proteolytic activity (PA) is determined according to the modified Anson method. PA of S. aureus (SaPA) is at a lower level than PA of P. aeruginosa (PsPA). Electrophoretic zymograms of proteases from these two bacteria are showed in figure 3. This research discovers 3 proteolytic bands from each bacterium, the proteases from P. aeruginosa have slow mobility, indicating that they have molecular mass higher than that of the S. aureus’s proteases. (1) ( 2) Figure 3. Proteinase from PsA (1), StA (2) (a) Gel without SDSC extracts (b) Gel with SDSC extracts Study PIA by Zymography This experiment is done as follows: enzyme solution loading on a polyacrylamide gel that contains polyphenol and substrate (casein). After electrophoresis, the gel is dyed with Amido Black 10B. All two polyphenol extracts from wood and SDSC significantly reduce activities of proteinase bands as shown on figure 3. Recent report shows methanol extract from Vietnamese sappan heart wood has the ability to induce cancer cell to apoptosis but has no inhibitory effect on caspase – 3, a cysteine protease in cell signaling pathway [23]. Polyphenol extracts might selectively target the bacteria proteases. Silicagel column chromatography In order to know whether flavonoid possessing the ability of inhibition on the studied proteases or not, the sappan wood flavonoid samples are fractionated on silicagel column chromatography, each collected fraction is subjected to running thin layer chromatography (TLC). A detected polyphenol spots are taken off separately and tested their PIA (by diffusion method). The results indicate that all five fractions obtained from the column possess PIA, but fractions I and fraction III are the strongest one. It means that the major flavonoids from C. sappan wood possess PIA. Figure 4. Re-chromatogram on TLC of Fraction I 1. Quercetin; 2.GV1; 3.GV2. Moreover, it is interesting to notice that fraction I shows a single band with a mobility equivalent to quercetin on TLC. Quercetin has been reported to be able to inhibit trypsin [28]. Therefore fraction I is subjected to re-chromatography with another solvent system, it generates 2 fractions named GV1 and GV2. GV1 has the mobility corresponding to that of quercetin on TLC (figure 4). PIA assay shows that GV1 exhibits PIA higher than that of GV2 (figure 5). UV-Vis analysis shows that, GV1 possesses 2 absorbance peaks at ultraviolet region below 280 nm (typical spectrum for isoflavone) (data not shown), while GV2 has one more peak at the region above 290 nm, resembling to quercetin (typical spectrum for flavonol, Phạm Thanh Kỳ et al. 2004) [14]. There have been published that C. sappan wood possesses homoisoflavonoid and its derivatives [10, 11], homoisoflavonoids are said to possess protease inhibitory ability [24], thus GV1 may be a homoisoflavonoid. For this reason, GV1 fraction is taken for structure analysis using NMR spectroscopy (this work is done in cooperation with Laboratory of Structural Chemistry, Institute of Chemistry, Vietnam Academy of Science and Technology). The result indicats that GV1 appear to be protosappanin A (fig. 6), a compound from homoisoflavonoid metabolism in C. sappan. 1 3 4 7 8 11 12 155 16 18 Figure 5. PIA from GV1 and GV2 factions 1-2: T(trypsin) + H2O, 3-4: T+ GV2; 5-6: T+GV1; 7-8 C(chymotrypsin) + H2O; 9-10: C+GV2; 11-12: C+GV1; 13-14: PsPA+H2O; 15-16: PsPA+GV2; 17+18: PsPA+GV1. CONCLUSION Among 26 samples from 20 medicinal plants belonging to 17 families have been studied, 21 aqueous samples of 16 plants have inhibitory activity against trypsin (TIA), chymotrypsin (ChIA) and extracellular proteinases from P. aeruginosa (PsIA). The obtained result of 80% studied samples possess PIA strongly supports our hypothesis that: the medicinal effects against pathogen of these plants are totally or partly due to the compounds innately existing in the plants that have inhibitory activity against proteases. Syzygium resinosum Gagnep has the highest TIA, PsIA, ChIA, about 11631, 8195, 33332 mIU/g fresh sample, respectively. TIA, PsIA, ChIA from the plants possessing high PIA (Syzygium formosum var. Syzygium formosum var. ternifolium (Wall) Matsam, Combretum quadrangulare Kurz., SDSC) are over 2400, 1500, 3500 (mIU/g fresh sample) respectively. 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Journal of Bioluminescence and Chemiluminescence, 24(5): 355-362. POLYPHENOL VÀ HOẠT TÍNH ỨC CHẾ PROTEASE TỪ MỘT SỐ CÂY THUỐC VIỆT NAM Nguyễn Minh Thắng, Phạm Thị Trân Châu Đại học quốc gia Hà Nội TÓM TẮT Chất ức chế proteinase có vai trò sống còn trong điều hòa các quá trình trao đổi chất và phát triển của sinh vật. Công trình này bước đầu thăm dò mối liên hệ giữa hoạt tính ức chế protease (PIA) và hàm lượng polyphenol trong một số cây thuốc Việt Nam thường dùng chữa mụn nhọt, mẩn ngứa. Từ 26 mẫu chiết trong nước của 20 loài thực vật thuộc 17 họ khác nhau, chúng tôi thu được 21 mẫu của 16 loài cây có hoạt tính ức chế trypsin (TIA), chymotrypsin (ChIA) và protease ngoại bào của Pseudomonas aeruginosa (PsIA). Kết quả cho thấy, dịch chiết từ các cây này ức chế chymotrypsin cao hơn hoạt độ ức chế các protease khác. Trong số các dịch chiết nước của các mẫu nghiên cứu, sắn thuyền (Syzygium resinosum Gagnep.) có PIA cao nhất (TIA, PsIA, ChIA lần lượt là 11631, 8195, 33332 mIU/g mẫu tươi). Các mẫu có PIA cao cũng có hàm lượng polyphenol cao. Dịch chiết ethanol polyphenol tổng số của hầu hết các mẫu có hàm lượng polyphenol cũng như PIA cao hơn so với dịch chiết nước. Sử dụng phương pháp điện di protease trên gel polyacrylamide (zymography) cho thấy các dịch chiết ethanol của gỗ vang và vỏ hạt chín ức chế cả 3 băng protease của P. aeruginossa và S. aureus. Sắc ký dịch chiết flavonoid từ gỗ vang qua cột silicagel, nhận được 5 phân đoạn có PIA. Sauk hi tái sắc ký, phân đoạn I tách thành 2 đỉnh, ký hiệu là GV1 và GV2, GV1 có PIA. Phân tích cấu trúc GV1 bằng phương pháp phổ NMR, cho thấy phân đoạn này có thể là protosappan A. Từ khóa: Pseudomonas aeruginosa, Staphylococcus aureus, Caesalpinia sappan, dịch chiết, ức chế protease. Ngày nhận bài: 22-10-2014