In vitro antioxidant and antibacterial activities of fractionated extracts and isolation of bioactive components from breynia vitis-Idaea (burm. f.) c. e. c. fischer leaves - Nguyen Thi Lan Huong

Journal of Science and Technology 54 (2C) (2016) 354-359 IN VITRO ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF FRACTIONATED EXTRACTS AND ISOLATION OF BIOACTIVE COMPONENTS FROM BREYNIA VITIS-IDAEA (BURM. F.) C. E. C. FISCHER LEAVES Nguyen Thi Lan Huong1, Nguyen Kim Minh Tam1, *, Huynh Ngoc Oanh1, Nguyen Thi Thu Huong2 1Hochiminh city University of Technology, VNU – HCM, 268 Ly Thuong Kiet St, D10, HCM 2Ginseng andMedicinal plants Research Centre, 41 Dinh Tien Hoang St, D1, HCM *Email: nguyenkmt@hcmut.edu.vn Received: 15 June 2016 ; Accepted for publication: 24 October 2016 ABSTRACT This study investigated the antioxidant and antibacterial activities of ethyl acetate, n- butanol and ethanol extracts of Breynia vitis-idaea (Burm.f.) C. E. C. Fischer leaves using in vitro assays;and isolated bioactive compounds from the fractioned extract which showed the best properties by column chromatography. All extracts showed significant radical scavenging activities and exhibited antibacterial activities against Enterococcus faecalis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). Ethyl acetate extracts showed the highest free radical scavenging capacity with the IC50 values of 99.55 and 94.66 μg/ml (in DPPH and ABTS assays, respectively) and exhibited MIC values of 1.5, 1 and 1 mg/ml against the three bacteria, respectively. In addition, from ethyl acetate extracts, one pure compound has been obtained and identified as 6-O-benzoylarbutin. Keywords:Breynia vitis-idaea, antioxidant activities, antibacterial activities,6-O-benzoylarbutin. 1. INTRODUCTION Breynia vitis-idaea (Burm.f.) Fischer (Euphorbiaceae) is an evergreen, large shrubwhich grows abundantly in Asian tropical regions, Africa, Australia and on some islands in the Pacific Ocean. In Vietnam, this plant has been discovered in many places, from northern regions down to Khanh Hoa, Lam Dong, Ba Ria – Vung Tau and Kien Giang (Phu Quoc island). Roots, leaves and barksof B. vitiss-idaeahave been used as medicinal ingredients[1, 2, 3]. Some studies have demonstrated that there are many bioactive compounds presenting in B. vitis-idaea such as glycosides, flavonoids, saponin, tannin etc.Hexane, ethyl acetate and methanol extracts of B. vitis-idaea showed significant radical scavenging activitiesattributed to the presence of phenolic compounds [4]. Methanol extracts also inhibited E. coli,S. aureus and Microsporum racemosum[5]. The ethanol and aqueous extracts of Breynia vitis-idaea leaves extracts proved that their anticancer activities might be due to alkaloids, glycosides, flavonoids and saponins [6]. In vitro antioxidant and antibacterial activities of fractionated extracts and isolation of bioactive ... 355 However, in-depth investigations on either chemical compositions or main activities of B. vitis- idaea remain limited in Vietnam. Therefore, based on medico-ethnobotanical knowledge about possible uses of B. vitis-idaea, this study aimed at obtaining and evaluating biological properties of various extracts from B. vitis-idaea leaves; isolating and determiningthe structure of one pure compound in the fraction that showed the best bioactivities. 2. EXPERIMENTS 2.1. Materials Fresh leaves of B. vitis-idaea were collected in Duc Hue District, Long An Province. The leaves wereshade dried for three days before being grounded into powder. 2.2. Method 2.2.1. Preparation of the extracts An amount of 20 g B. vitis-idaea leaf powder was extracted three times, each time with 100 ml of ethanol 96 % in 24 hours at room temperature. The ethanol extract (EtOH) was removed fats by shaking with n-hexane (n-Hex); then the undissolved part was extracted with different organic solvents in increasing polarity order [ethyl acetate (EtOAc) and n-butanol (n-BuOH)] to get respective extracts. 2.2.2. DPPH radical scavenging assay This assay was conducted according to the method prescribed by Rao (1996) with some modifications [7]. An amount of 100 μl DPPH solution (6 mM) and 100 μl of various concentrations of the extracts or standard solution (ascorbic acid) were added into 2800 μl of methanol. The mixtures were incubated at 37 oC for 30 min. Absorbance of each solution was measured at 517 nm. The radical scavenging activity of samples was calculated as the percent DPPH radical scavenging effect. IC50 value is calculated by GraphPad Prism via the base line of inhibition percentage (base line established from 6 different concentrations). 2.2.3. Scavenging of ABTS radical cation This assay was based on the method of Re et al. [8]. ABTS+ was produced by mixing 7 mM ABTS with 2.45mM K2S2O8. The mixture was stored in the dark at room temperature for 12 – 16hours. The ABTS radical solution was diluted with ethanol to adjust its absorbance to 0.7 ± 0.02 at 734 nm. To determine the scavenging activity, 0.2 ml of various concentrations of the extracts or the standard were mixed with 3ml ABTS solution. The absorbance was measured after 20 minutes at 734 nm. The 50 % inhibitory concentration (IC50) values of leaf extracts were calculated by GraphPad Prism via the base line of inhibition percentage (base line established from 6 different concentrations). 2.2.4. Antibacterial bioassay Antibacterial activities of extracts against Escherichiacoli ATCC 25922, methicillin- resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC Nguyễn Thị Lan Hương, Nguyễn Kim Minh Tâm, Huỳnh Ngọc Oanh ... 356 27853, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Salmonella TyphimuriumATCC 14028 were assessed by agar dilution method and minimum inhibition concentrations (MICs) of each extract were determined [9]. 2.2.5. Isolation, refinement and structure determination of the isolated compound The EtOAc extract (5g) was chromatographed on a column (40 mmx 600 mm) of silica gel (15μm, Merck, Germany) which was developed with a mixture of n-hexane and an increasing proportion of ethyl acetate: 0.6 l n-Hex; 1.2 l n-Hex: EtOAc (95: 5); 1 l n-Hex: EtOAc (90: 10); 3.8 l n-Hex: EtOAc (80: 20); 1.8 l n-Hex: EtOAc (60: 40); 0.7 l n-Hex: EtOAc (40: 60); 1.4 l n- Hex: EtOAc (20: 80); 0.9 l EtOAc.Elution with n-Hex: EtOAc (20: 80) afforded three fractions. Fraction showing the least number of bandsby TLC wasselected for concentration. The resulting solid (0.5178 g) was repeatedly crystallized from ethyl acetate to yield 0.2665 g white crystalline powder (marked F18). The purecrystal was tested by one-direct nuclear magnetic resonance (1H-NMR, 13C-NMR, and DEPT) and two-direct nuclear magnetic resonance (COSY, HSQC and HMBC) spectrometry on NMR Bruker Avance II to find its specific structural characteristics, and measured mass spectrometry on Agilent 1200 Series coupled to MS detector to determine its molecular mass. These experiments were conducted atthe Central Laboratory for Analysis of Hochiminh City University of Science. 3. RESULTS AND DISCUSION 3.1. Bioactivities of fractionated extracts From the ethanol extract, two smaller extractsalso obtained were ethyl acetate (EtOAc) and n-butanol (n-BuOH). These two extracts together with ethanol extract (EtOH) were tested with antimicrobial and antioxidant activities. The results were presented in Table 1 and 2. Antimicrobial activity of the three extracts was relatively low. Both the EtOH and EtOAc extracts could only inhibit Gram-positive bacteria (E. faecalis, S. aureus and MRSA) but could not resist Gram-negative bacteria (E. coli, Sal. typhi and P. aeruginosa) whereas the n-BuOH extract didnot have any activity on all tested bacteria. MIC values of the EtOAc extract on Gram-positive bacteria were two-fold lower than that of the EtOH; and this demonstrated that the activity of EtOAc extract was the highest. Table 1. MIC value of the extracts on tested bacteria (mg/ml). Extract E. coli E. faecalis Sal. typhi P. aeruginosa S. aureus MRSA EtOH - 3 - - 2 1.5 EtOAc - 1.5 - - 1 1 n-BuOH - - - - - - All of the three extracts of B. vitis-idaea showed significant antioxidant activity in two different methods of antioxidant assays tested (Table 2). The highest DPPH scavenging activity was observed in EtOAc (IC50 99.55 ± 1.26 μg/ml) followed by EtOH (109 ± 1.66 μg/ml) and n- In vitro antioxidant and antibacterial activities of fractionated extracts and isolation of bioactive ... 357 BuOH (370.3 ± 2.48 μg/ml). Similar trend was observed in scavenging activity of ABTS (EtOAc > EtOH > n-BuOH). The activities of these extracts were concentration-dependent. Table 2. Antioxidant activities of the fractionated extracts from B. vitis-idaea. Extracts IC50 (µg/ml) DPPH ABTS EtOH 109 ± 1.66 115.5 ± 0.88 EtOAc 99.55 ± 1.26 94.66 ± 0.69 n-BuOH 370.3 ± 2.48 181.1 ± 0.68 Vitamin C 16.36 ± 1.46 16.21 ± 2.48 From the above data, it could be inferred that EtOAc extract had the highest bioactivity because it contained the most bioactives. This fraction was used in isolation and purification experiments to get pure compound. 3.2. Structure determination of the F18 by spectrometry From the analysis of NMR spectra of fraction F18, the predicted formula was C19H20O8. The structure of F18 coincided with the structure of eximine (6-O-benzoylarbutin, a polyphenolic glycoside) (Fig. 1) reported to decrease tyrosinase activity and melanin content with little evidence of cytotoxicity. 13C NMR (500 MHz, DMSO), δ ppm: 63.8 (s, C-6’), 69.7 (s, C-4’), 72.7 (s, C-3’), 73.1 (s, C-2’), 75.9 (s, C-5’), 100.8 (s, C-1’), 114.8 (d, C-2, C-6), 117.0 (d, C-3, C-5), 128.2 (d, C-3”, C- 5”), 128.6 (d, C-2”, C-6”), 129.2 (s, C-1”), 132.8 (s, C-4”), 149.6 (s, C-4), 151.7 (s, C-1), 165.0 (s, C-7”) 1H NMR (500 MHz, DMSO), δ ppm: 3.26 (m, 3H, H-2’, H-3’, H-4’), 3.69 (t, 1H, H-5’), 4.26 (dd, 1H, H-6’-B), 4.60 (dd, 1H, H-6’-A), 4.73 (d, 1H, H-1)’, 5.28 (s, 3H, OH), 6.56 (d, 2H, H-3, H-5), 6.84 (d, 2H, H-2, H-6), 7.56 (t, 2H, H-3”, H-5”), 7.69 (t, 1H, H-4”), 7.97 (d, 2H, H- 2”, H-6”), 9.006(s, 1H, OH) [s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet (denotes complex pattern)] Having measured ESI-HRMS, the spectrogram of F18 displayed peak 399.1061, corresponding to the piece of [M + Na]+, in proportion to molecular mass of 399.1050 (C19H20O8Na). Figure 1. Chemical structure of 6-O-benzoylarbutin. Nguyễn Thị Lan Hương, Nguyễn Kim Minh Tâm, Huỳnh Ngọc Oanh ... 358 Breynia vitis-idaea was used for curing body aches and skin diseases. Ethanol and ethyl acetate extract of the leaves of this plant recorded inhibitory activity against E. faecalis, which causes stomachache, S. aureus and MRSA, which cause skin infections, with MIC under 3 mg/ml. With regard to E. coli, Sal. typhi and P. aeruginosa, all of the extracts did not resist them at concentration of 5 mg/ml which was too high in comparison with a common antibiotic. It was interesting to note that ethanol and ethyl acetate extract was only inhibitory to Gram-positive bacteria, and this indicated the presence of more than one active principle which could act on Gram-positive and Gram-negative bacteria separately. This difference can be explained by the mechanism of effect of antibiotics on Gram-negative and Gram-positive bacteria. The distinction in the wall structure of Gram-positive and Gram-negative bacteria may be the cause resulting in the dominant effect of antibiotics or antibacterial compounds. The antioxidant capacity of the plant extract largely depends on both the constituent of the extract and the test system. There are a lot of factors that may influence this capacity. Therefore, more than one type of antioxidant capacity measurement should be performed to take into account of the various mechanisms of antioxidant actions[4]. One of the widely used chromogens that has received considerable use in chemical methods of antioxidant detection is the stable free radicals, DPPH and ABTS•+ (both of them are foreign to biological systems), in the DPPH and ABTS assay, respectively. The results of the ABTS assay should be comparable to results found in the DPPH assay and may be viewed as confirmation of the DDPH assay [10]. In comparison with extract without enzyme, enzymatic extracts either with DPPH or ABTS methods recorded moderate antioxidant activities. The significant radical scavenging potential of B. vitis-idaea extracts may be due to hydroxyl groups presenting in the phytochemicals. Strong antioxidant activities and medicinal functions of B. vitis-idaea make it a promising source of natural antioxidants in food and pharmaceutical industries. 6-O-benzoylarbutin is also called eximine, a common compound which was extracted from some plants belonging to Breynia and Protea genus. Eximine was one of the acylated arbutin derivatives which were desmontrated that they had antioxidant and antibacterial activity [11]. 4. CONCLUSION The extracts obtained from B. vitis-idaea leaves showed significant antioxidant activities and had inhibitory effect on the growth of E. faecalis, S. aureus and MRSA. From EtOAc extract, 6-O-benzoylarbutin was isolated. Acknowledgements. 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FISCHER LEAVES Nguyễn Thị Lan Hương1, Nguyễn Kim Minh Tâm1, *, Huỳnh Ngọc Oanh1, Nguyễn Thị Thu Hương2 1Trường Đại học Bách khoa, Đại học Quốc gia TpHCM, 268 Lý Thường Kiệt, Q10, TpHCM 2Trung tâm Sâm và Dược liệu, 41 Đinh Tiên Hoàng, Q1, TpHCM *Email: nguyenkmt@hcmut.edu.vn Bằng các thử nghiệm invitrochúng tôi đánh giá hoạt tính kháng oxy hóa và kháng khuẩn của các cao chiết ethyl acetate, n-butanol và ethanol từ lá cù đề Breynia vitis-idaea (Burm.f.) C. E. C. Fischer leaves using;đồng thời chúng tôi tiến hành tách chiết hoạt chất từ cao chiết cho hoạt tính sinh học cao nhất bằng sắc ký cột. Tất cả các phân đoạn cao chiết đều có hoạt tính kháng oxy hóa và kháng các chủng vi khuẩn Enterococcus faecalis, Staphylococcus aureusvà Staphylococcus aureuskháng methicillin. Cao ethyl acetatecho IC50đối với gốc do DPPH và ABTS lần lượt là 99,55 và 94,66 μg/ml và MIC trên 3 chủng vi khuẩn trên lần lượt là 1,5; 1;và 1 mg/ml. Từ cao ethyl acetate, chúng tôi đã tách được 1 hoạt chất tinh khiết và xác định được chất đó là 6-O-benzoylarbutin. Keywords:Breynia vitis-idaea, hoạt tính sinh học,6-O-benzoylarbutin.