Applied Mechanics and Materials Vols. 754-755 (2015) pp 1040-1044 © (2015) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMM.754-755.1040
Submitted: 05.01.2015 Accepted: 05.01.2015
Antibacterial activity of different biomass components of Cerbera odollam and their potential to be used as new preservative for wood based products. M.H.M. Amini1,a*, R. Hashim2,a, N.S. Sulaiman2,b, O. Sulaiman2,c, S.F. Sulaiman3,a, F. Abood4,a, F. Kawamura5,a, R. Wahab1,b, M. Mohamed1,c, M.S.M. Rasat1,d 1
Faculty of Earth Science, Universiti Malaysia Kelantan, 17600 Jeli, Kelantan, Malaysia
2
Division of Bio-resource, Paper and Coatings Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia. 3
School of Biology, Universiti Sains Malaysia, 11800 Penang, Malaysia.
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Faculty of Forestry, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
5
Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686, Japan. E-mail:
[email protected],
[email protected],
[email protected],
[email protected],
[email protected],
[email protected],
[email protected], h
[email protected],
[email protected],
[email protected]
d
Key words: Cerbera odollam, antibacterial, borer, preservative
Abstract. Cerbera odollam’s tree parts were extracted with methanol and further fractionated using n-hexane, ethyl acetate and ethanol, followed by antibacterial assay against Bacillus subtilis, Bacillus licheniformis, Escherichia coli and Pseudomonas aeruginosa. Hexane soluble parts from flower, fruit, leaf, wood, bark and ethyl acetate soluble part from bark showed antibacterial activity against Bacillus subtilis. Hexane soluble parts from leaf and bark and ethyl acetate soluble parts from wood showed antibacterial activity against Bacillus licheniformis. All fractions appeared ineffective on Escherichia coli and Pseudomonas aeruginosa. Screened extracts were analysed using FTIR. Experiments were continued with impregnation of methanolic extracts of Cerbera odollam into Melamine-urea formaldehyde (MUF) particleboards, Phenol-resorcinol formaldehyde (PRF) particleboards and solid wood samples followed by exposure to wood-boring beetles and natural decaying. Introduction Cerbera odollam or also known as pong pong tree were originally found in mangrove swamps but widely grown in parks, gardens and roadside as a shade tree. Belongs to the poisonous Apocynaceae family, the fruits are apple look alike. The seed is protected by a thick fibrous layer under the smooth outer layer [1]. Cerbera odollam is well known for several traditional uses in some countries. Research by Gaillard et al. [2] found out that this so called suicide tree responsible for many plant poisoning cases in Kerala, India due to high cardiac glycoside (cerberin) content especially in the seed. The seeds are widely used as rat poison. The cytotoxic cardenolide glycoside from the seeds of cerbera odollam showed cytotoxic activities against oral human epidermoid carcinoma, human breast cancer cell and human small cell lung cancer [3]. Cerberin and cerberoside from the kernels of Cerbera odollam were also being tested on several animals to test for their toxicity. Injected into frog’s lymph sac, cerberoside slows down the frog’s heart rate besides premature beats were observed while experiment on cats showed a marked rise of blood pressure, arrhythmia and sudden circulatory collapse [4] while ethanolic extract of Cerbera All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 58.26.188.235-09/02/15,11:13:30)
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odollam’s leaves on albino Swiss mice found that it inhibits mice’s spontaneous movement [5]. Rahman et al. [6] had worked on the effect of extracts from the seed of Cerbera odollam on larvae of Culex quinquefasciatus and Aedes aegypti. N-butanol extracts of the seed showed positive effect on the larvae mortility where percentage of mortility increased with increasing concentration of the extract. Recently, the usage of artificial chemicals as wood preservative draws concern on human health and environmental safety. Many researches have been done in order to find a more environmental friendly wood preservative. It was proven that extractives from tropical timbers have potential to increase the durability of wood [7]. Many researches had shown that extracts from plants could be utilized as an antibacterial agent. The poisonous Cerbera odollam’s tree parts were also believed could be utilized as wood preservative to replace or reduce the usage of conventional wood preservatives. Antibacterial assay against Bacillus subtilis, Bacillus licheniformis, Escherichia coli and Pseudomonas aeruginosa were done and effect of impregnation of extractives from different parts of Cerbera odollam on decay resistance of wood against wood boring beetles and natural decaying agents were investigated besides its suitability to be utilized as wood preservative. Materials and Experimental Details Extraction and fractionation process for antibacterial assay Samples from different parts of Cerbera odollam were collected around the Universiti Sains Malaysia, Penang, Malaysia. Parts from the plant collected include the fruit, seed, leaves, flower, wood and bark. Samples were dried and pulverized in the grinding machine. The moisture content was measured based on oven dry weight of sample. Extraction and fractionation procedures were carried out based on Kawamura et al., [8]. The samples were extracted using methanol for 1, 2 and 3 hours in sequence. The extracted solution was filtered through cotton wool and dried using the rotary evaporator. After methanol extraction, the same sample was extracted with hot water, filtered and freeze dried. Extract from methanol extraction was fractionized successively with n-hexane, ethyl acetate and ethanol to give four extracts that were n-hexane soluble, EtOAc soluble, EtOH soluble and insoluble following method by Kawamura et al. [8] with slight modification. Antibacterial activity assay Extractives from the different parts of biomass components of Cerbera odollam were exposed to bacteria to determine its antimicrobial activity. Four types of bacteria were used in this study that includes Bacillus subtilis (ATCC 21332), Bacillus licheniformis (ATCC 14580), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Antibacterial activity assays of the extractives were carried out by the disc diffusion assay method on agar medium [9]. Extracts fractions that showed inhibition zone in antibacterial assay were analysed using FTIR [10]. Particleboard testing Samples of fruit, wood, bark, leaf, seed and flower of the tree were cut into smaller pieces and left air-dried. Dried samples were extracted using methanol by immersion method for 10 days. Extractive solutions were filtered using cotton wool and dried using rotary evaporator and ready to be used [11]. Rubberwood blocks (Havea brasiliensis) were obtained from Kedah, Malaysia, cut into spesific sizes for borer and soil burial test and soaked in methanol for 10 days to remove as much as possible extractives from them. Wood blocks were dried in oven at 1000C to remove solvent and moisture before impregnating the extractives of Cerbera odollam into it by immersion of wood sample in 1% extractive solution following method by Kamdem [12] with slight modification. Particleboards were made with 1% addition of Cerbera odollam extractives [13]. Susceptibility test against wood-boring beetle was done following method in BS EN 47 [14] with
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modification. Soil burial test was done following method stated in BS 1982-2 [15] with necessary modifications. Results and discussions Antibacterial assay The effect of extracts on the growth of Bacillus subtilis and Bacillus licheniformis showed that hexane soluble part from bark had the highest inhibitory effect on Bacillus subtilis. Hexane soluble part from bark has strongest inhibitory effect on Bacillus licheniformis bacteria followed by hexane soluble part from the flower. Ethyl acetate soluble part from bark, hexane soluble part from leaf, hexane soluble part from wood and hexane soluble part from fruit, showed less antibacterial activity on the bacteria. There was no antibacterial activity observed on Escherichia coli and Pseudomonas aeruginosa. This could be because of insufficient concentration of extract or Gram negative bacteria characteristic that are more resistant to antimicrobial agent than the Gram-positive bacteria. One of the several unique characteristics of Gram-negative bacteria is the outer membrane of the cell that is responsible for protecting the bacteria from destruction of the inner membrane or cell wall (peptidoglican). The outer membrane is built up of semipermeable barrier to the uptake of antibiotic, antibacterial or biocides [16]. The P. aeruginosa is naturally resistant to a large range of antibiotics and may demonstrate additional resistance after unsuccessful treatment through mutation of their porin which is opening on the outer membrane surface that control movement of substance, in and out. Results of the experiment could be affected by many factors for instance the accumulation of lipid by the bacteria cell in action of resistance and the ability of some bacteria to decompose antibacterial agents. Same species of bacteria could resist differently towards an antibacterial agent. The functional groups present in the extracts with antibacterial activity were detected using FTIR. Analysis showed that the extracts from Cerbera odollam contains several carbonyl groups, appears as carboxylic acids, aldehydes, ketones, esters and amides. Hydroxyl containing groups and nitrogen containing groups were also found in the extracts. The lipophilic character of hydrocarbon skeleton and the hydrophilic character of functional groups of the compounds play main importance in the antimicrobial action of plant extract with the sequence of phenols with the highest activity followed by aldehydes, ketones, alcohols, esters and the least antimicrobial activity is the hydrocarbons. Exposure to wood-boring beetles and soil burial test For wood-boring beetle decay resistant test, there is no significant result could be determined between different extractivesas shown in Table 1. Comparison between MUF particleboard, PRF particleboard and solid wood samples impregnated with extractives and their weight loss after exposure to the insect showed that wood panels impregnated with any extractives always more resist to attack compared to untreated, control sample. For MUF particleboards, only control board was significantly different from other samples while for PRF particleboards, samples treated with commercial preservative and control board were significantly different from other particleboards. Results of the expeiment depends largely on the distribution of wood borers in the test chamber. Samples were placed randomly to ensure the attacks of insects occured evenly among the samples. Solid wood sample impregnated with methanolic extract from leaf showed highest decay resistant with only 4.53% decay while control sample of solid wood showed highest degradation with 12.47% decay. Samples of particleboards were less destroyed because the adhesives used in board making were also have some toxic effect on the insects.
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Table 1: Wood-boring beetles exposure test and soil burial test results Testing results Particleboard Impregnated Extractives type Wood borers decay, % Soil burial decay, % Leaf 5.98±0.03a 19.59±14.89a Fruit 5.47±0.17a 9.85±3.30b Wood 5.32±0.55a 9.19±2.47c MUF Bark 5.53±0.33a 11.24±3.47d particleboards Flower 5.69±0.26a 5.46±2.66e Seed 5.76±0.17a 6.05±2.11f Commercial preservative 5.13±0.58a 1.45±0.80g Control sample 6.75±0.06ab 23.05±26.71h Leaf 6.10±0.29a 24.34±14.67a Fruit 5.57±0.07a 34.74±11.35b Wood 5.70±0.12a 29.41±0.55c Bark 5.64±0.06a 28.00±26.32d PRF Flower 5.88±0.27a 18.41±16.94e particleboards Seed 5.77±0.14a 23.39±6.55f Commercial preservative 4.84±0.81b 7.16±7.49g Control sample 7.62±0.41c 55.22±22.74h Leaf 4.53±0.82a 9.94±0.44a Fruit 6.42±1.33b 11.87±0.67a Wood 10.50±11.51 c 10.08±0.82b 11.54±10.91d 10.28±0.33c Solid wood Bark samples Flower 6.13±3.13eb 10.49±5.02d Seed 7.47±3.76f 10.23±0.32ebc Commercial preservative 6.14±0.73gbe 7.41±0.37f Control sample 12.47±12.32h 12.19±3.82g Values with different letters within same column are significantly different at alpha value of 0.05 Results of soil burial test are also shown in Table 8 where it could be determine that wood panels impregnated with commercial preservative have most less weight loss compared to control samples which possessed the highest wood decay. There is extraordinary weight loss in control sample of PRF board, probably because the soil site where it was buried has more natural wood decaying agents such as insects like termites or anything else. It was also the reason why the standard deviation were so high where results of the experiment depends on too many uncontrollable factors. Solid wood sample were more stable because it was less affected by weather like rain which particleboards absorbed more moisture than solid wood making it less resistant to decay.
Conclusions 1. Hexane soluble parts from flower, fruit, leaf, wood, bark and ethyl acetate soluble part from bark showed antibacterial activity against Bacillus subtilis. 2. The hexane soluble parts from leaf and bark and ethyl acetate soluble parts from wood also showed antibacterial activity against Bacillus licheniformis. 3. All of the extracts and fractions showed no antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. 4. Control sample of solid wood showed highest degradation with 12.47% decay in wood-boring beetles resistance test.
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Acknowledgement We would like to acknowledge to Ministry of Science, Technology and Innovation, Malaysia (MOSTI) for the grant support and Universiti Sains Malaysia for Fellowship grant in order to complete this research. Reference [1] A.V. Tung, in: Know the Wayside Trees of Malaysia: Broadleaves 2, Volume 4, Media Kelisa. Selangor, Malaysia (2005) [2] Y. Gaillard, A. Krishnamoorthy, F. Bevalot, in: Journal of Ethnopharmacology 95, 123–126 (2004) [3] S. Laphookhieo, S. Cheenpracha, C. Karalai, S. Chantrapromma, Y. Rat-a-pa, C. Ponglimanont, K. Chantrapromma, in: Phytochemistry 65, 507 – 510 (2004) [4] K. K. Chen, F. A. Steldt, in: Journal of Pharmacology & Expert Therapy 76, 167 (1942) [5] M. T. T. Hien, C. Navarro-Delmasure, T. Vy, in: Journal of Ethnopharmacology 34, 201-206 (1991) [6] R. A. Rahman, A. Kadri, I. M. Said, in: Proceeding of Tropical Natural Resource Symposium, Kuching, Sarawak, Malaysia (1993) [7] G. Nemli, E. D. Gezer, S. Yıldız, A. Temiz and A. Aydın, in: Bioresource Technology 97, 2059–2064 (2006) [8] F. Kawamura, S. Ohara, A. Nishida, In: Holzforschung 58, 189 – 192 (2004) [9] S. Dash, L. K. Nath, S. Bhise, N. Bhuyan, in: Tropical Journal of Pharmaceutical Research 4 (1), 341-347 (2005) [10] J. H. Muyonga, C. G. B. Cole, K. G. Duodu, in: Food Chemistry 86, 325–332 (2004) [11] G. A. Ayoola, A. D. Folawewo, S. A. Adesegun, O. O. Abioro, A. A. Adepoju-Bello, H. A. B. Coker, in: African Journal of Plant Science 2 (9), pp. 124-128 (2008) [12] D. P. Kamdem, in: Forest Product Journal 44 (1), 30 – 32 (1994) [13] BS EN 47, in: British Standard 1 – 24 (2005) [14] BS 1982-2, in: British Standards 1 – 10 (1990) [15] R. E. W. Hancock, in: Clinical Infectious Diseases 27, S93–S99 (1998) [16] A. R. Koroch, H. R. Juliani, J. A. Zygadlo, in: Flavours and Fragrances, pp. 87-115 (2007)
