Antioxidant and antibacterial activities of agarwood (Aquilaria malaccensis Lamk.) leaves

Transcription

1 Antioxidant and antibacterial activities of agarwood (Aquilaria malaccensis Lamk.) leaves Hadi Hendra, Sukarti Moeljopawiro, and Tri Rini Nuringtyas Citation: AIP Conference Proceedings 1755, 144 (216); View online: View Table of Contents: Published by the American Institute of Physics Articles you may be interested in Inhibitory effect of wild mango (Mangifera foetida L.) extract on seed germination of Cynodons dactylon (L.) Pers. AIP Conference Proceedings 1744, 29 (216); 1.163/ Phenotypic traits of Cucumis melo L. cv. Tacapa and commercial melon cultivars based on multilocation and multiseason trials AIP Conference Proceedings 1744, 24 (216); 1.163/ Metabolic profiling of endophytic bacteria from Purwoceng (Pimpinella pruatjan Molkend) root and antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa AIP Conference Proceedings 1744, 63 (216); 1.163/ Molecular identification and optimization culture conditions of amylase producing bacteria isolated from green algae in the coast side of Southern Sea, Yogyakarta, Indonesia AIP Conference Proceedings 1755, 2 (216); 1.163/ Primer design and in silico analysis using CLUSTALW and MUSCLE for L-arabinose isomerase (araa) gene detection in thermophilic bacteria AIP Conference Proceedings 1755, 147 (216); 1.163/ The effect of early posthatch local feed in pectoralis muscle of Jawa Super chicks (Gallus gallus domesticus) AIP Conference Proceedings 1755, 143 (216); 1.163/

2 Antioxidant and Antibacterial Activities of Agarwood (Aquilaria malaccensis Lamk.) Leaves Hadi Hendra 1, Sukarti Moeljopawiro 1 1, a) and Tri Rini Nuringtyas 1 Laboratory of Biochemistry, Faculty of Biology, Universitas Gadjah Mada Jl. Teknika Selatan, Sekip Utara, Yogyakarta, Indonesia Corresponding author: a) tririni@ugm.ac.id Abstract. Several factors involve in the synthesis of secondary metabolites in plants, including leaves maturity. The objectives of this study were to compare the antioxidant and antibacterial activities of agarwood Aquilaria malaccensis Lamk. leaves at different stages of maturity as well as to identify the group of compounds responsible to the corresponding activities. Young and old leaves of agarwood were used. Leaves were powdery dried and extracted using three different polarity solvents including chloroform, methanol and water using Soxhlet apparatus. The potential extract was fractionated using Vacuum Liquid Chromatography (VLC). Antioxidant activity was analysed using 2,2-diphenyl-1- picrylhydrazyl (DPPH) while antibacterial activity was evaluated using paper disc diffusion assay. Thin Layer Chromatography (TLC) followed by spraying with specific reagents was applied to identify groups of bioactive compounds in the potential fraction. The results showed that the methanol extract of the old leaves was the most potential extract with an IC5 value of ± 1.49 μg/ml and the combined fraction of 1:3 chloroform: methanol and 1% methanol showed the highest antioxidant activity with IC ± 1.43 μg/ml. The chloroform extract of old leaves showed the highest antibacterial activity on S. aureus and E. coli with a diameter of inhibition zone of 1.83 mm and 9.92 mm respectively at concentration extract of mg/ml. While the most potential fraction as antibacterial on S. aureus and E. coli was chloroform fraction that showed 3 times higher activity than the crude extract. The fraction with highest antioxidant activity contained phenolic and flavonoid, while the potential antibacterial fraction contained alkaloid and terpenoid. Old agarwood of A. malaccensis leaves showed higher potency as an antioxidant and antibacterial than the young leaves. INTRODUCTION The use of plants as herbal medicines is associated with the content of secondary metabolites. Several factors involve in the production of secondary metabolites in plants, including leave age. The distribution pattern of secondary metabolites is affected by the type of organ and growth stage of plants [1]. However, people usually do not consider the growth stage when they use plants in traditional medicines, especially for herbal drinks. In several areas in Indonesia, agarwood (Aquilaria malaccensis Lamk.) leaves have been popular as herbal drinks. Aside from that, the resin of agarwood has also been used for scents, perfumes, and cosmetics. Nowadays, the use of agarwood, not limited to resin, but has been extended as aromatherapy, soaps, preservatives, pharmaceuticals with several bioactivities such as antiasthmatic, antimicrobial, face and skin treatments, malaria drug [2], analgesic, antipyretic, antioxidant [3] and anti-hyperglycemia [4] as well as nerve and gastrointestinal stimulants [5, 6], However, the scientific information of these bioactivities of agarwood leaves in Indonesia is still very limited. A study on the secondary metabolites of agarwood leaves revealed that the leaves contained alkaloids, flavonoids, triterpenoids, steroids and saponins [7]. Phenolics, anthocyanins, and flavonoids in plants has highly related to the antioxidant activity [8]. Thus, it is most likely that the high efficacy of agarwood leaves as herbal drinks is closely related to its antioxidant activity. Furthermore, these compounds together with saponins and alkaloids have a proven record as antimicrobial agents in many other plants [9]. Advances of Science and Technology for Society AIP Conf. Proc. 1755, ; doi: 1.163/ Published by AIP Publishing /$

3 Nowadays, studies to search natural antibacterial compounds have increased steeply since the use of synthetic antibacterial drugs in the long period have been associated with the emergence of drug-resistance bacteria. Thus, it is necessary to get alternative drugs treatment of bacterial infections from natural compounds. In Indonesia, there was no report on the study of antioxidants and antibacterial activities of young and old leaves of A. malaccensis. The purposes of this research were studying the antioxidant and antibacterial activities of different polarity leaves extracts, fractionation of potential extract, identification group of secondary metabolites that act as antioxidant and antibacterial in the most potential fraction and analysis of the antioxidant and antibacterial activities in the different growth stage of agarwood leaves. MATERIALS AND METHODS Materials Young and old leaves of agarwood A. malaccensis were collected from Bogor Botanical Garden, West Java, Indonesia. Young leaves of agarwood determined by the light green colour of the leaves, soft texture and the first to the seventh leaf from the shoot, whereas the old leaves used to have dark green colour, hard and tough texture, and the leaves located after the seventh leaf from the shoot. Pathogenic bacteria used were Staphylococcus aureus IFO13276 and Escherichia coli IFO331. Preparation and Extraction of Agarwood Leaves After washing with water, leaves were air-dried in shade for 7 days and continued drying using the oven at 4 C until reaching their constant weight. Subsequently, leaves were powdered using a blender. A total of 1 g of leave powder was consecutively extracted using chloroform (pa. Merck) and methanol (pa. Merck) (15 ml for each solvent) using soxhlet apparatus. The water extract was obtained by boiling the residue from the previous extraction until the volume reduced to half of initial volume. All crude extracts were evaporated to dryness using water bath at 6 C. The dark brown sticky extracts were weighed and stored at 4 C for further use. 1,1-diphenyl-2-picrylhydrazyl (DPPH) Test Antioxidant activity was conducted according to the methods of Pyrzynska and Pekal [1]. To 1 ml extract solution, 1 ml of.1 mm DPPH (Sigma- Aldrich) in methanol was added and mixed until homogeneous. The mixture was incubated at room temperature for 3 minutes in the dark. Blank solution was prepared by mixing 1 ml of methanol and 1 ml DPPH. The absorbance of the mixture was measured at 517 nm using UV-Vis spectrophotometer (Genesys). Vitamin C (Sigma-Aldrich) was used as positive control. The antioxidant activity was determined as the blank absorbance subtracted by the absorbance of extract and divided by the absorbance of the blank. Antibacterial Activity Preparation of bacteria suspension The bacteria suspension was prepared by mixing each isolates S. aureus and E. coli in 1 ml of.9 % sterile saline. The bacterial turbidity was adjusted to have a similar absorbance with.5 Mc-Farland solution at.1 OD λ625 nm to reach 1 8 cfu / ml of bacteria suspension. Paper Disc Diffusion Test. A sterile petri dish containing 15 ml of Meuller Hinton Agar (MHA) (Merck) media was inoculated with.1 ml of the bacterial suspension and incubated for 24 hours at 37 C. Six crude extracts including chloroform, methanol and water from both old and young leaves as well as solvents and positive control were evaluated in the series concentration of 15- mg/ml. Each paper disc with a diameter of 6 mm was placed on media after previously dipped into the corresponding extract. Positive control was 3 μg/ml ampicillin (Roche) and the paper disc dipped in 144-2

4 positive control was placed in the centre of the medium. The antibacterial activity of the tested extracts was shown by a clear zone of inhibition around the application point. Fractionation of Potential Extract The potential extracts for antioxidant and antibacterial were fractionated by vacuum liquid chromatography (VLC) on silica gel 6 F254 (Sigma) using a mixture of different solvents ranging from non-polar to polar including chloroform, ethyl acetate, methanol, and water. The fraction having a similar profile on TLC were combined and subjected again to the antioxidant and antibacterial assay using a similar method for the crude extract but at a lower concentration range of 5-25 μg/ml for antioxidant assay and mg/ml for antibacterial assay. Identification of the Group of Compounds in the Active Fractions The most potential fractions for antioxidant and antibacterial activities were analysed using TLC and then visualized under visible light, UV light at 254 nm and 366 nm as well as spraying using various specific reagents detection including Anisaldehyde, Lieberman-Burchard, Dragendroff and FeCl 3 to identify the groups of compounds. Data Analysis All experiments were conducted in triplicate. Antioxidant activity was analyzed by probit analysis to determine the IC 5 of extracts and fractions. While antibacterial data of extracts and fractions were analyzed by ANOVA (Analysis of Variance) and DMRT (Duncan's Multiple Range Test) posthoc analysis. RESULT AND DISCUSSION Agarwood Leaves Extraction The type of solvent affects the amount of extracts obtained [11]. Non-polar and polar solvents have different capabilities and effectiveness in extracting secondary metabolites [12]. According to Godwill et al. [13], water and methanol are the best solvents for extraction because of the number and types of compounds extracted more than other solvents such as ethyl acetate and hexane. In this study, the highest yield of extraction was methanol extract of old agarwood leaves at the percentage of 9.2%. While the lowest extract yield was water extract of young agarwood leaves at the percentage of 1.6%. Thus, in term of the yield of extract, the best solvent for extraction was methanol, followed by chloroform and water. This result applied to both young and old agarwood leaves. TABLE 1. Yields of agarwood leave extraction using three solvents at different leaves age Weight (g) Percentage (%) Extracts Dry Young Leaves Old Leaves Young Old leaves Simplicia Extract Extract leaves Chloroform Methanol Water The old agarwood leaves contain more secondary metabolites than the young ones. These seem to be a general result in plants as also mentioned by Achakzai et al. [1] who reported a similar distribution pattern of secondary metabolites in a different type of organ and age of the several plants in India. Each plant has a specific distribution pattern for different types of secondary metabolites which differ at every stage of development and its organs. These results were consistent with the TLC monitoring data of extract which showed that old leaves extract have more spots than the young leaves extracts. It indicates that the more amount of the spot showed that the more variety of secondary metabolites and affect its bioactivity. These results were observed in all type of extracts

5 Antioxidant Activity of Agarwood Leaves Antioxidant activity of crude extract The results showed that old leaves extract had higher antioxidant activity than the young leaves. Furthermore, there was a positive correlation between increasing the concentration of extract to the increasing activity of free radicals scavenging was observed in this research (Fig. 1). This result is in line with previous research by Dewi [14] showing methanol extract of old leaves of A. malaccensis had the highest antioxidant activity than methanol extract of young and mature leaves. According to growth-differentiation balance hypothesis (GDBH), parts or organs in plants with a longer period of growth will have more resources available for defense, so it has a higher level of defence compounds / secondary metabolites than the younger organs [15]. Thus the high antioxidant activity of old leaves extract may be caused by the higher content of secondary metabolites of old leaves compared to young leaves. The methanol extract of old agarwood leaves showed the highest antioxidant activity compared to chloroform as well as water extract at IC 5 value of ± 1.49 μg / ml. Water extract of young leaves and chloroform extract for both old and young leaves showed the low antioxidant activity with IC 5 > 15 μg / ml (Table 2). A similar study using A. malaccensis leaves from Malaysia also showed that the methanol extract was observed to have the highest antioxidant activity compared to hexane, dichloromethane, and ethyl acetate extracts [7]. Methanol extracts may include phenolic, alcohols, sugars or glycosides [16] these compounds have been associated with the antioxidant activity of plant materials. FIGURE 1. Free radical scavenging activity of young and old agarwood leaves extracts TABLE 2. IC5 value of agarwood leaves extracts and vitamin C Extracts IC5 (μg/ml) Chloroform extract of young leaves >15 Methanol extract of young leaves ± 2.12 Water extract of young leaves >15 Chloroform of old leaves >15 Methanol extract of old leaves ± 1.49 * Water extract of old leaves ± 1.91 Vitamin C 2.12 ±.38 *= extract with lowest IC5 value Fractionation of Potential Extract The methanol extract of old leaves was further fractionated using several combination of solvent at different polarity. Seven fractions were obtained and based on their TLC profile, several fractions with similar profile were combined resulting 3 combined fractions (Fig. 2)

6 1 2 3 FIGURE 2. A representative diagram of TLC profiles obtained from the methanol extract of the old agarwood leaves. The numbers designated with arrows represent the combined fractions. Antioxidant activity of Fractions The combined fractions III (CFIII) had the highest antioxidant activities at IC 5 value of ± 1.43 μg/ml. The combined fractions I (CFI) and II (CFII) had IC 5 higher than 25 μg/ml. The antioxidant activity of the most active fraction showed a positive correlation with the concentration of extract (Fig. 3) The high antioxidant activity of the CFIII may correlate with the polarity of the eluents used in the fractionation. The CFIII had higher polarity compared to other fractions. In general, the natural antioxidants from plants derive from phenolic compounds especially phenylpropanoids, anthocyanins and flavonoids which have polar character [17]. The methanol extract of A. malaccensis leaves contained various secondary metabolites such as alkaloids, flavonoids, steroids, saponins and tannins that potential as an antibacterial, antioxidant, anti-inflammatory, analgesic, and antimalarial drug [18]. FIGURE 3. The correlation between type and concentration of the combined fraction with the reduction percentage of free radicals Antibacterial Activity of Agarwood leaves Antibacterial activity of crude extract All agarwood leaves extracts had antibacterial activities on S. aureus and E. coli but differ in strength (Table 3). Old leaves extract gave bigger inhibition zone than young leaves extract. These results were observed in both S. aureus and E. coli in all type of extracts (Table 3). Thus, this result underlined the antioxidant activity result that the old leaves were more potential than young leaves. Statistical analysis proved that leaves age, type of extract and concentration affected antibacterial activities on S. aureus as well as E. coli. Chloroform extract of young and old leaves had higher antibacterial activities compared to other types of extracts. Increasing concentrations of extract led to increasing in the diameter of inhibition zone. These results are consistent with a previous study reporting inhibition zone diameter of hexane, dichloromethane, 144-5

7 and methanol extracts derived from leaves and bark of Aquilaria crassna increased by the increase of concentrations of extracts against several gram-positive bacteria and negative test [19]. Order of antibacterial activity from highest to lowest of the leaves extracts was the extract of chloroform followed by methanol and water extracts. The high antibacterial activity in chloroform extract was possible because chloroform can extract various components which act as antibacterial compounds including terpenoids, alkaloids, and flavonoids better than methanol and water [2, 21]. TABLE 3. Antibacterial activity of agarwood Aquilaria malaccensis Lamk. leaves extract against S. aureus and E. coli Concentration Diameter of Inhibition Zone (mm) Extracts (mg/ml) Staphylococcus aureus Escherichia coli a 7.42 a 7.92 Chloroform extract b 7.58 a 8.75 of young leaves bc 8.42 b 1. c 8.75 b Methanol extract of young leaves a 7. a 7.17 b 7.33 b 6.75 a 7. a 7.5 b 7.67 b Water extract of young leaves a 6.75 ab 6.83 b 7. b 6.58 a 6.83 a 7. a 7. a Chloroform extract old leaves a 9.17 b 9.67 bc 1.83 c 7.75 a 9.25 b 9.67 b 9.92 b Methanol extract of old leaves a 6.92 a 7. ab 7.42 b 6.83 a 7.33 ab 7.75 bc 7.92 c Water extract of old leaves a 6.92 a 7.8 a 7.17 a 6.67 a 6.92 ab 7.8 ab 7.67 b Description: The same letters within a column indicates not significantly different by ANOVA at significance level of 5% Antibacterial Activity of fraction Fractionation of old leaves chloroform extract was conducted using four eluents. Based on the TLC profiles observed under visible light, UV light λ 254 nm and λ 366 nm, four fractions were combined into two fractions (Fig. 4). The antibacterial activity assay showed that combined fractioned I (ChFI) had higher antibacterial activity than the combined fraction II (CFII) on both S. aureus and E. coli (Table 4). Chloroform fraction had an inhibition zone diameter of 9.58 mm and 9.8 mm on S. aureus and E. coli respectively at a concentration of 1 mg/ml. This activity was three times higher than the crude chloroform extracts. The concentration of 1 mg/ml was not significantly different from the concentration of 5 mg/ml in the test against S. aureus. So, the effective concentration of ChFI against S.aureus was 5 mg/ml (Table 4)

8 1 2 FIGURE 4. A representative diagram of TLC profiles obtained from the chloroform extract of the old agarwood leaves. The numbers designated with arrows represent the combined fractions. TABLE 4. The antibacterial activity of fraction Fractions Concentration Diameter of Inhibition Zone (mm) (mg/ml) Staphylococcus aureus Escherichia coli Chloroform fraction a a 7.83 ab 8,71 bc 9.58 c 6,75 a 6,83 a 8. b 9.8 c Combined fraction b a 6.83 a 7.88 b 8.5 b 6.42 a 6.92 ab 7.38 b 8.13 c Description: The different letters within a column indicate significantly different treatments by ANOVA at a significance level of 5% Identification Group of Compounds of Most Potential Fractions Identification group of compounds of the potential fractions showed that the CFIII which was the potential fraction for antioxidant activity contained two groups of compounds, phenolic and flavonoids compounds and negative for terpenoids. Whereas the ChFI which was the potential fraction for antibacterial activity contained alkaloid and terpenoids compound and negative for terpenoids, phenolic and saponin compounds (Table 5)

9 Fractions Combined fraction III (CFIII) Chloroform fraction (ChFI) TABLE 5. The result of identification group of potential fraction Group of Amount of Racing Spray Reagent Positive Description Compound Spot Factor (Rf) Control Phenolic 1,29 FeCl 3 Gallic acid Positive, deep greenblack spot color in visible light Flavonoid 1,47 Sitroboric Quercetin Positive, yellow spot in λ 366 nm Terpenoid - Anisaldehyde Tymol Negative, not detected Sulfuric acid Alkaloid 1.53 Dragendorf Quinine Positive, orange spot in visible light Terpenoid 3.22 ;.33 Anisaldehyde Tymol Positive, red-violet in ;.59 Sulfuric acid visible light Phenolic - FeCl3 Gallic acid Negative, not detected Saponin - Liebermann- Saponin Negative, not detected Burchard CONCLUSION The methanol extract of the old leaves of Aquilaria malaccensis Lamk. had the highest antioxidant activity whereas the chloroform extract of old agarwood leaves showed the highest antibacterial activity on S. aureus and E. coli. The potential fraction of methanol extract of old leaves showed a slightly lower IC 5 compared to the crude extract. The potential antibacterial fraction derived from the chloroform extract of old leaves had three times higher activity than the crude extract. A group of compounds acting as antioxidants was phenolic and flavonoids while the group of compounds that contribute as antibacterial compounds was alkaloids and terpenoids. Old leaves of agarwood were more potential as antioxidant and antibacterial than young leaves. REFERENCES 1. A. B. K. Achakzai, P. Achakzai, A. Masood, S. A. Kayani and R. B. Tareen, Pak. J. Bot., 41, (9). 2. A. Sofyan, A. Sumadi, A. Kurniawan, dan A. Nurlia, Pengembangan dan peningkatan produktivitas pohon penghasil gaharu sebagai bahan obat di Sumatera dalam Laporan Hasil Penelitian Program Insentif Peningkatan Kemampuan Peneliti dan Perekayasa (Kementerian Kehutanan Balai Penelitian Kehutanan, Palembang, 21). 3. J. Sattayasai, J. Bantadkit, C. Aromdee, E. Lattmann and W. Airarat, J. Ayurveda Integr. Med. 3, (212). 4. R. Pranakhon, P. Pannangpetch, and C. Aromdee, J. Sci. Technol. 33, (211). 5. S. A. Siran, Perkembangan Pemanfaatan Gaharu, Pengembangan Teknologi Produksi Gaharu Berbasis Pemberdayaan Masyarakat Sekitar Hutan edited by in S.A. Siran, M. Turjaman (Pusat Penelitian dan Pengembangan Hutan dan Konservasi Alam, Bogor, 21), pp Gusmaliana, B. Wiyono, and T. Waluyo, Buku Ajar Analisis Hayati, (Penerbit Buku Kedokteran EGC, Jakarta, 21). 7. A.W.N Huda, M.A.S Munira, S.D. Fitrya and M. Salmah, Pharmacognosy Res. 1, (9). 8. G. Cao E. Sofic and R. L. Prior, Free Radicals Biol. Med. 22, (1997). 9. J. C. Escalona-Arranz, P. R. Renato, U.L Imilci, C. P. Miladis Isabel, J. R. Amado, and L. J. Pharmacogn Mag. 6, (21). 1. K. Pyrzynska, A. Pękal, Anal Methods 5, (213). 11. G. J. Gil-Ch avez, A. Jos e, Villa, J. Fernando, Ayala-Zavala, J. B. Heredia, D. Sepulveda, E. M Yahia and Gonz alez-aguilar. Comprehensive Reviews in Food Science and Food Safety 12, (213). 12. N. P. Tambellini, V. Zaremberg, R.J. Turner, and A.M. Weljie, Metabolites 3, (213). 13. E. A. Godwill, N. Paul, N. J. Chidubem, O. T. Innocent and E. B Chinenye, International Journal of Pharmacognosy and Phytochemical Research 5, (213)

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