Antioxidant, antibacterial and alpha amylase inhibitory activity of different fractions of sonneratia apetala bark extract - Dao Van Tan

TAP CHI SINH HOC 2014, 37(1se): 54-60 DOI: 10.15625/0866-7160/v37n1se. ANTIOXIDANT, ANTIBACTERIAL AND ALPHA AMYLASE INHIBITORY ACTIVITY OF DIFFERENT FRACTIONS OF Sonneratia apetala Bark EXTRACT Dao Van Tan*, Mai Nhat Thuy Hanoi National University of Education, Hanoi, *daotanvn@yahoo.com ABSTRACT: Sonneratia apetala was imported into Vietnam in August 2003 and planted in XuanThuy National Park (Nam Dinh province). Samples of barks were collected and extracted with methanol and fractioned with ethyl acetate and 0.1% HCl in methanol using Sep-pak plus cartridges. Total polyphenol content of crude extract and the fractions was determined, and their antioxidant and antibacterial activity and Aspergillus niger α-amylase inhibitory activity was tested. Crude extract possessed the highest total polyphenol content (10973.2±2453.5 µg GAE/ml), followed by methanol-HCl fraction (124.6±10.4 µg GAE/ml) and ethyl acetate fraction (96.6±13.0 µg GAE/ml). Crude extract showed the highest antioxidant activity (IC50=3.4±0.3 µg/ml), equal to 0.86 folds of acid ascorbic activity using 1, 1-diphenyl-2- picrylhydrazyl (DPPH) scavenging assay, followed by methanol-HCl fraction (IC50=5.7±0.2 µg/ml) and ethyl acetate fraction (IC50=8.7±0.8 µg/ml). No antibacterial activity of ethyl acetate fraction was observed while the activity of crude extract against B. subtilis, S. aureus and that activity of methanol-HCl fraction against E. coli, B. subtilis were observed. The higher inhibition of Aspergillus niger α-amylase also was observed for the fractions of methanol-HCl (IC50=6.6±0.9 µg/ml) and ethyl acetate (IC50=16.9±1.0 µg/ml) compared to that of crude extract (IC50=300.4±66.0 µg/ml). Keywords: Sonneratia apetala, amylase inhibitor, antioxidant, antibacterial, Sep-pak plus cartridge. INTRODUCTION Recent studies have shown that plants, including mangrove plants were a potential source to provide high bioactivity compounds such as antioxidant, antibacterial compounds. Azuma et al. (2002) [1] have reported terpenoids and some fatty acids in flowers of mangrove plants. Beula et al. (2012) [2] have evaluated antiviral, antioxidant and toxicological activity of mangrove plants from South East coast of India. In our previous studies, we have reported some potential bioactivities of Derris trifoliata, Acanthus ilicifolius, Pluchea pteropoda, and Ipomoea pes-caprea from mangrove plants of Xuan Thuy National Park [11, 19, 22, 23]. Besides, Bhutkar et al. (2012) [3] and Subramanian et al. (2008) [20] have suggested that finding inhibitors of carbohydrate hydrolyzing enzymes, such as α-amylase and α-glucosidase might be a potential trend in the control of diabetes. Ramkumar et al. (2010) [17] have studied in vitro alpha-glucosidase and alpha-amylase enzyme inhibitory and reported positive effects of Andrographis paniculata extract and andrographolide. Sonneratia apetala, a mangrove species belonging to the family Sonneratiaceae, was imported into Vietnam in August 2003 and planted in Xuan Thuy National Park (Nam Dinh province). The mangrove plant is thought to be a favorite species for expanding mangrove areas. Studies on Sonneratia apetala in Vietnam are focused on tolerance, growth and other biological characteristics [24]. There were a few studies on bioactivities of S. apetala both in the World and in Vietnam. Bobbarala et al. (2009) [4] have tested antimicrobial activity of hexane, chloroform and methanol extracts S. apetala of mixed areal parts. Milon et al. (2012) [12] have tested S. apetala bark extract in the inhibition of antibiotic resistant bacteria and phytopathogens. However, evaluation of bioactivity (such as amylase inhibitory activity) of fractions using Sep-pak plus cartridges has not been reported yet. Therefore, in the present study, total polyphenol content, antioxidant, antibacterial, and Aspergilllus niger α-amylase inhibitory activity of methanolic extract and different fractions using Sep-pak cartridges for searching for novel bioactive products from mangrove plants have been investigated. MATERIALS AND METHODS Ascorbic acid, agar, and DPPH were purchased from Sigma- Aldrich Co. Other chemicals were made in India. Sonneratia apetala (Buch.- Ham.) bark was collected from the mangrove forest of XuanThuy National Park. After collection, the samples were washed by tap water then air dried for 96 hours in shading condition. The dried samples were ground into powder in liquors nitrogen and stored in an airtight container. Extraction Five grams (5 g) of the powder were soaked in 50 ml of methanol for 72 hr, and then filtered with Whatman no.1 filter paper to obtain the first filtrate. The filter residue was soaked in 25 ml methanol for 3 hr and then filtered to obtain the second filtrate. Both filtrates were combined. Six milliliters of the combined filtrate was vapored to obtain crude solid extract, and then dissolved in 5 mL distilled water (crude extract). Six milliliters of the combined filtrate were loaded into double Sep-pak plus cartridges and eluted with ethyl acetate and then 0.1% HCl in methanol according to material described by Kim & Lee (2002) [9]. The elusions were vapored to obtain solid extracts, and then each extract was dissolved in 5 mL distilled water to obtain fractions ethyl acetate and 0.1% HCl in methanol, respectively. Determination of total polyphenol Total polyphenol content was determined using reagent phenol Folin-Ciocalteu [8]: 100 µl of the samples at various concentration were mixed with 10 µl of reagent Folin-Ciocalteu for 5 min and then 100 µl Na2CO3 7% were added, being incubated for 90 min in dark. The mixture then was measured at absorbance at 750 nm using spectrometer (Biotek, USA). Gallic acid was used as the standard. Antibacterial tests Four bacteria were supplied by the Department of Biotechnology and Microbiology, Hanoi National University of Education, being purchased from National Institute of Hygiene and Epidemiology, namely Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Samonella tiphimurium. One hundred microliters (100 μL) of extract and the fractions were subjected to antibacterial assay using the agar well diffusion method described by Murray et al. (1995) [14]. The culture medium was MPA. For the positive control, 100 μL of 0.04% chloramphenicol for the negative control, 100 μL of distilled water were used instead of crude extract or the fractions. The discs were incubated at 4ºC for 8 hours for diffusing, and then were transferred to the condition of 30ºC for 24 hours. The zones of inhibition were measured with antibiotic zone scale in mm. The experiment was carried out in triplicates. Determination of inhibition of α-amylase α-amylase (EC 3.2.1.1) extracted from Aspergilllus niger was used for enzyme test. An equal volume of enzyme and inhibitor at different dilutions were mixed and incubated for 10 min. The reaction mixture containing 120 µl of 1% starch in pH 5.0 and 20 µl of enzyme with 20 µl of inhibitor (test) or distilled water (control) was incubated for 30 at 30°C. Enzyme activity was determined using soluble starch as a substrate according to Geriacheva’s method [13]. Fifty microliters (50 µl) of reaction mixture were mixed 950 µl of iodine solution (0.01%I and 0.1% KI in 0.1N HCl). Enzyme activity was defined as the quantity of alpha-amylase that will hydrolyze soluble starch at the rate of one gram per hour at 30°C. Absorbance of each reaction was measured at 656 nm using spectrometer (Biotek, USA). Inhibitory activity of α-amylase was determined using value EC50. Determination of antioxidant activity The antioxidant activity was determined by 1,1- diphenyl-2-picrylhydrazy (DPPH) radical scavenging activity as described by Ibeh (2013)[6]. One milliliter of the reaction mixture consisting of 0.95 mL of 0.3 mM DPPH in ethanol was mixed with 0.05 mL of samples. Ascorbic acid at dilution series was used for the standard curve. For blank, 0.95 ml of ethanol was mixed with the samples. For control, 0.95 mL of 0.3 mM of DPPH in ethanol was mixed with 0.05 mL of samples. After incubation at room temperature for 30 min in dark condition, the amount of DPPH remaining was determined by measuring absorbance at 517 nm using spectrometer (Biotek, USA). Mean values were obtained from triplicate experiments. Data access Data was accessed using software SPSS. Each assay was repeated at least three times. RESULTS AND DISCUSSION Total polyphenol Total polyphenol of crude extract, ethyl acetate and methanol-HCl fractions was determined (table 1). The crude extract possessed the highest content of total polyphenol (10973.2 µg GAE/ml (gallic acid equivalence). There was no significant difference in total polyphenol content between fractions ethyl acetate and methanol-HCl. The fractions possessed low contents of total polyphenol (95.6 and 124.4 µg GAE/ml for fraction ethyl acetate and methanol-HCl, respectively). Data also suggested that there was a large loss of polyphenol amount during separation. Table 1. Total polyphenol content of crude extract and the fractions of ethyl acetate and methanol-HCl Total polyphenol content (µg GAE/ml) Total polyphenol content (µg GAE/mg) Crude extract fraction 10973.2±2454.5a 685.8±153.3a Ethyl acetate fraction 96.6±13.0b 290.1±39.7b Methanol-HCl 124.6±10.4b 249.2±20.8b The analytical data are the mean of triplicate (mean±standard division). Different letters (a,b) indicate differences of values in the same column at P<0.05 using Tukey test. Table 2. Antibacterial activity (inhibition zone mm) of crude extract and the fractions of ethyl acetate and methanol-HCl E. coli B. subtilis S. aureus S. tiphimurium Crude extract - 3.0±1.0a+ 3.0±0.8a - Ethyl-acetate fraction - - - - Methanol-HCl fraction 6.3±4.0b 5.7±1.2b* - - The analytical data are the mean of triplicate independent measurements (mean±standard division). Different letters (a,b) indicate differences of values in the same row and different symbols (+,*) indicate differences of values in the same column at P<0.05 using Tukey test. Antibacterial activity Data on antibacterial activity was shown in table 2. Antibacterial activity of positive control, 0.04% chloramphenicol, against test bacteria was 15.6 mm and no inhibition zones of negative control were observed. Activity of the crude extract against Bacillus subtilis, Staphylococcus aureus was observed with inhibition zones of 3 mm. No antibacterial activity of ethyl acetate fraction was observed while that activity of methanol-HCl fraction against E. coli, B. subtilis was observed with inhibition zones of 6.3 and 5.7 mm, respectively. The difference in antibacterial activity among the crude extract and the fractions suggested the difference in property of compounds in different fraction. Bobbarala et al. (2009) [4] have investigated antimicrobial activity of aereal parts of S. apetala against 15 microorganisms and showed that hexane, chloroform and methanol extracts showed a good activity against all the pathogens, with zone of inhibition from 10 to 22 mm. Those authors indicated that these extracts were active against E. coli (15.7 mm) and S. aureus (18.7 mm). Our findings are in agreement with those study results on activity of barks against both E. coli and S. aureus in spite of a low activity. Bark extracts with carbon tetrachloride had antibacterial effects against E.coli (8 mm), B. subtilus (11 mm), V. parahaemolyticus (7 mm) and S. aureus (7 mm) whereas extracts with n-hexane and chloroform did not have the effects [12]. In comparison with other mangrove species, such as Acanthus ilicifolius, Ipomoea pes-caprae and Pluchea pteropoda grown in Xuan Thuy National Park [11, 19, 22] using Soxhlet extraction, antibacterial activity of Sonneratia apetala bark against the same bacteria had no better effects. The low antibacterial activity of Sonneratia apetala in the present study might be due to the use of different extraction methods. Table 3. DPPH radical scavenging activity of crude extract and the fractions of ethyl acetate and methanol-HCl IC50 (µg/ml) Folds of acid ascorbic activity Crude extract 3.4±0.3a 0.87±0.64 Ethyl acetate fraction 5.8±2.3b 0.54±0.36 Methanol-HCl fraction 8.7±0.8c 0.35±0.23 The analytical data are the mean of triplicate independent measurements (mean ± standard division). Different letters (a,b,c) indicate differences of values in the same column at P < 0.01 using Tukey test. Table 4. Inhibition of Aspergilllus niger α-amylase by crude extract and the fractions of ethyl acetate and methanol-HCl IC50 (µg/ml) Crude extract 300.4±66.0a Ethyl acetate fraction 16.9±1.0b Methanol-HCl fraction 6.9±0.9c The analytical data are the mean of triplicate independent measurements (mean±standard division). Different letters (a,b,c) indicate differences of values at P<0.05 using Tukey test. Antioxidant activity Antioxidant activity was determined by 1,1- diphenyl-2-picrylhydrazy (DPPH) radical scavenging activity. The data is shown in table 3. Crude extract showed the highest activity (IC50=3.4 µg/ml) equal to 0.86 folds of acid ascorbic activity while this activity of the fractions ethyl acetate and methanol-HCl was observed to be 5.8 and 8.7 µg/ml, equal to 0.54 and 0.35 folds of acid acetic activity, respectively. In a recent study, Qusti et al. (2010) [16] have shown that a compound, which had DPPH radical scavenging activity at IC50 value less than 10 µg/ml was classified into high activity. The antioxidant activity of crude extract and the fractions of Sonneratia apetala in the present study were observed to be high. Inhibition of Aspergillus niger α–amylase To compare inhibition of Aspergillus niger α-amylase by crude extract and the fractions, the value IC50 was compared. The data is shown in table 4. The fraction methanol-HCl exhibited the highest inhibition (IC50=6.9 µg/ml) followed by the fraction ethyl acetate (IC50=16.9 µg/ml) and crude extract (IC50=300.4 µg/ml). Tamil et al. (2010)[21] in their study on α-amylase inhibitory activity of Phyllanthus amarus extracts against porcine pancreatic amylase in vitro, have shown that the ethanol and hexane extracts of P. amarus exhibited appreciable α-amylase inhibitory activity with an IC50 values 36.05±4.01 μg/mL and 48.92±3.43 μg/mL, respectively. Kumar and Sudha (2012) [10], studied α-amylase inhibitory activity of four seaweeds, showing that extracts from all 4 species exhibited α-amylase inhibitory activity. The value IC50 was observed to be 67 µg/ml, 60 µg/ml, 83 µg/ml, and 82 µg/ml for Ulva lactuca, Sargassum polycystum, Gracilaria edulis, and Gracilaria corticata, respectively. In comparison with α-amylase inhibitory activity of other plant species [7, 15, 18], α-amylase inhibitory activity of ethyl acetate and methanol-HCl fractions was quite high. Therefore, our study supports usage of extract of Sonneratia apetala bark in ethnological medicines for control of diabetes. CONCLUSION Sonneratia apetala bark collected from XuanThuy National Park possessed a high total polyphenol content. There was, however, a loss of polyphenol during fraction. Rude extract and the fractions possessed slight antibacterial activity against tested bacteria. Crude extract and the fractions showed the high antioxidant activity. 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HOẠT TÍNH CHỐNG OXY HÓA, KHÁNG KHUẨN VÀ ỨC CHẾ ALPHA AMYLASE CỦA CÁC PHÂN ĐOẠN KHÁC NHAU CHIẾT XUẤT TỪ VỎ THÂN BẦN KHÔNG CÁNH (Sonneratia apetala Buch.-Ham.) Đào Văn Tấn, Mai Nhật Thùy Trường Đại học Sư phạm Hà Nội TÓM TẮT Cây Bần không cánh (Sonneratia apetala) nhập từ Myanmar về Việt Nam vào năm 2003 và được trồng tại Vườn Quốc gia Xuân Thủy, Nam Định. Mẫu vỏ thân bần không cánh lấy từ rừng ngập mặn Xuân Thủy được xử lí và chiết bằng methanol. Sau đó dịch chiết methanol được phân đoạn bằng dung môi ethyl acetate và methanol có HCl 0,1% sử dụng Sep-pak plus cartridge. Hàm lượng polyphenol tổng số, hoạt tính chống oxy hóa, sự ức chế sinh trưởng thực vật, khả năng ức chế α-amylase Aspergilllus niger của các phân đoạn của chiết xuất thân bần không cánh được xác định. Dịch chiết thô methanol chứa hàm lượng polyphenol tổng số lớn nhất (10973,2±2453,5 µg GAE/ml), phân đoạn methanol-HCl (124,6±10,4 µg GAE/ml) và phân đoạn ethyl acetate (96,6±13,0 µg GAE/ml) có hàm lượng phenol tổng số thấp hơn. Hoạt tính chống oxy hóa của dịch chiết thô vỏ thân bần không cánh khá cao, giá trị IC50 cho quét gốc tự do DPPH là 3,4±0,3 µg/ml, tương đương 0,86 lần so với hoạt tính của acid ascorbic, các phân đoạn đoạn ethyl acetate và methanol-HCl có hoạt tính chống oxy hóa kém hơn dịch chiết thô với giá trị IC50 lần lượt là 5,7±0,2 µg/ml; 8,7±0,8 µg/ml. Không phát hiện có hoạt tính kháng của phân đoạn ethyl acetate đối với các chủng vi khuẩn thử nghiệm, trong khi dịch chiết thô có hoạt tính kháng đối với B. subtilis, S. aureus và phân đoạn methanol-HCl có hoạt tính kháng đối với E. coli, B. subtilis. Hoạt tính ức chế α-amylase Aspergilllus niger mạnh nhất đối ở phân đoạn methanol-HCl (IC50 =6,6±0,9 µg/ml), tiếp đến phân đoạn ethyl acetate (IC50 =16,9±1,0 µg/ml) trong khi dịch chiết thô thể hiện khả năng ức chế kém nhất (IC50 =300,4±66,0 µg/ml). Từ khóa: Sonneratia apetala, chống oxy hóa, kháng khuẩn, Sep-pak plus cartridge, ức chế amylase. Ngày nhận bài: 22-10-2014