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LSP99 Biological Activities of Leaf and Bark from Aquilaria crassna Pierre (Gaharu) H. Alimon1, N. Mohd Arriffin2, S.S. Syed Abdul Azziz2, R. Ibrahim2, F. Mohd Jaafar3 and M.A. Mohd Sukari4
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Department of Biology, 2Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia 3 Department of Chemistry, Faculty of Applied Sciences, Universiti Teknologi Mara, 40450 Shah Alam, Selangor, Malaysia 4 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Email: hasimah@fsmt.upsi.edu.my

Abstract Crude extracts of leaves and bark of Aquilaria crassna Pierre (Gaharu) using three different solvents: hexane, dichloromethane and methanol, had been screened for antimicrobial activities toward four bacteria; Pseudomonas aeruginosa, Bacillus spizizenii, Staphylococcus aureus and Shigela flexneri. The results showed that Staphylococcus aureus was the most susceptible strain to all six crude extracts, in which the highest inhibition was exhibited by the methanol extract of the leaf. The least susceptible strain was Pseudomonas aeruginosa of which only the bark methanol extract showed the inhibition zone.
Key words: Aquilaria crassna, Thymelaeceae, antimicrobial

Abstrak Ekstrak daun dan batang Aquilaria crassna Pierre (Gaharu) menggunakan tiga jenis pelarut berbeza; heksana, diklorometana dan metanol, telah disaring untuk aktiviti antimikrob terhadap empat bakteria; Pseudomonas aeruginosa, Bacillus spizizenii, Staphylococcus aureus dan Shigela flexneri. Keputusan menunjukkan Staphylococcus aureus adalah strain yang paling mudah direncat oleh kesemua enam ekstrak mentah, dengan perencatan tertinggi ditunjukkan oleh ekstrak metanol daun. Manakala, strain yang paling kurang direncat adalah Pseudomonas aeruginosa, iaitu hanya ekstrak metanol batang menunjukkan zon perencatan.
Kata kunci: Aquilaria crassna, Thymelaeceae, antimikrobial

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Introduction Gaharu or agarwood (aloeswood, eaglewood) is a resinous, fragrant and highly valuable heartwood of Aquilaria species from the Thymelaeceae family. There are altogether 16 species of Aquilaria. The different species was found in different country. However, the best known species that produce the gaharu resin are Aquilaria malaccensis and Aquilaria crassna. A. crassna is an evergreen tree of medium-size and can reach to a height of 1520m and a diameter at breast height of 40-50cm [1]. One of the traditional usages of gaharu is for the production of incense which is used in important religious ceremonies, rituals and meditation. Gaharu is widely used in traditional medicine as sedative, analgesic and digestive [2,3]. The gaharu oil is highly demanded for its use in perfumes and toiletry products such as shampoo and soap. Recently phytochemical and pharmacological studies on gaharu have increased. However, previous scientific work had only focused on the oil of the gaharu instead of other parts of the plant such as the leaves and bark. According to Zhou et. al. [4] the leaves of gaharu were used in China for the treatment of trauma-related diseases such as fractures and bruises. Unfortunately relatively little is known about the chemical constituents of the gaharu leaves. Dash et. al. [5] reported the antibacterial activity of the aqueous and methanol extracts of the Aquilaria agallocha Roxb leaf and bark dry powders against four bacteria; Shigella flexneri, Bacillus brevis, Pseudomonas aeruginosa and Bacillus subtilis using agar well method. The zone of clearance around each well was measured after the incubation period. The results showed that there were inhibition zones around the well and confirmed the antimicrobial activity of the respective extract. A clear inhibition towards Shigella flexneri and Pseudomonas aeruginosa with range 14-18 mm was observed from the aqueous extracts of both leaf and bark produced. While the methanol extract of leaf had shown the highest inhibition zone with 19 mm against B. subtilis, the methanol extracts of bark did not show any inhibition. It clearly proved that gaharu trees have potential as an antimicrobial agent. Nonetheless, further investigation has to be done to confirm the constituents of the tree that also act as antimicrobial compounds. Materials and Methods The leaves and barks of Aquilaria crassna was collected from Kajang, Selangor, Malaysia on July 2008. Plant material (leaves and barks) were air dried before use (Fig. 1 and Fig. 2). Approximately 1 kg each of the leaves and bark were extracted successively using hexane, dichloromethane and methanol at room temperature. The solvent extracts were filtered and evaporated under reduced pressure to give their crude extracts. A total of six plant extracts were tested for antimicrobial activity.

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Fig. 1: Leaves of A. crassna

Fig. 2: Bark of A. crassna

Preparation of Stock Solutions of Plant Extracts. Stock solutions of the plant extracts were prepared by dissolving 200 mg of each plant extract in 1 ml of sterilized distilled water. The mixture was vortexed to ensure that the extracts were homogeneous. The working stock solutions were protected from light by covering the bottle with aluminium foil. Plant extracts stock solutions were used in disc diffusion test and broth dilution method. Culture Media. The bacterial cultures used in this study were Bacillus subtilis (B. spizizenii) (ATCC 6633), Staphylococcus aureus (ATCC 1026), Pseudomonas aeruginosa (ATCC 10145) and Shigella flexneri (ATCC 12022). The bacteria were purchased from Choice Care Sdn. Bhd. The test organisms were purified and maintained on slant agar kept at 4o C until further usage. Preparation of Media. Nutrient agar was used as the media for bacteria enumeration. Nutrient broth was also used for the generation of exponential culture of each organism. Nutrient Agar. 15 g nutrient agar was suspended with 1 liter of distilled water in a Duran bottle. The media was dissolved by fast cooking in the microwave and autoclaved for about 15 minute at 121oC. The sterilized melted agar was poured into sterile Petri dishes in the laminar flow for the agar plate preparation. Nutrient Broth. 13 g nutrient broth was suspended with 1 liter of distilled water. The media dissolved by fast cooking in the microwave. 5 ml of melted broth were pipetted into test tube and sterilized by autoclaving for about 15 minutes at 121oC. Screening for Antibacterial Activity. The antibacterial activity of the extracts on the microorganisms was determined by using the disc diffusion and broth tube dilution methods. Disc Diffusion Method. The disc diffusion method was used to measure the rate of inhibition in growth of bacteria by different concentrations of the plant extract on paper disc. The volumes of extract used were 20 l, 30 l, 40 l and 50 l from the 200 mg/ml of each plant extract stock solution. The discs were allowed to dry in the laminar flow before they were placed on top of the agar. The 24 hours broth culture of each test bacterium species was aseptically introduced and spread on the surface of sterile nutrient agar using a sterile cotton swab. The sterile paper discs (6 mm) impregnated with extract were placed on the cultured plates and sterile forceps were used to gently press down each disc to ensure complete contact with agar surface. A disc with distilled water alone served as negative control. The plates were incubated at 370C for 24-48 hours. All experiments were performed in triplicate and each experiment was reproduced a minimum of three times and all these procedures were carried out aseptically. Determination of Antibacterial Properties. Reading of the inhibition zones was done at 24 and 48 hour-intervals. The antibacterial activity was interpreted from the size of the diameter of zone inhibition measured to the nearest mm as it is observed from the clear zone surrounding the disc. The zone of inhibition is measured from the edge of the disc to the edge of the growth. It is measured on the undersurface of the plate without opening the lid.

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Results and Discussion Six extracts were tested on four bacteria; Pseudomonas aeruginosa, Bacillus spizizenii, Staphylococcus aureus and Shigela flexneri, and observed after 24 and 48 hours. The results shown below are based on the solvent used: dichloromethane (Table 1 and Table 2), hexane (Table 3 and Table 4), and methanol (Table 5 and Table 6). The dichloromethane extracts of the leaf and bark of A. crassna showed almost similar inhibition zones against three out of four bacteria tested for both incubation times (Table 1 and Table 2); Bacillus spizizenii, Staphylococcus aureus and Shigela flexneri. An increase in the inhibition zones occurred as the concentration of the extracts increased. However, there was not much difference in the size of the inhibition zones to the incubation times for all three bacteria. Nevertheless, the dichloromethane extracts showed highest inhibition growth against Staphylococcus aureus whereas no inhibition activity was shown against Pseudomonas aeruginosa at the two incubation times. Table 1: Inhibition zones of dichloromethane extract of A. crassna after 24 hours incubation Microorganism Plant Part Concentration of plant extract (mg/ml) 6 8 Inhibition zone (mm) 8.67 0.58 8.270.06 9.00 0.00 8.330.06 Control DW

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Gram-positive Pseudomonas aeruginosa Shigella flexneri Gram-negative Bacillus spizizenii

L B L B

8.33 0.58 8.100.10

9.33 0.58 8.500.00

L 8.00 0.00 8.33 0.29 8.50 0.00 9.00 0.00 B 8.000.00 8.000.00 8.000.00 8.000.00 L 9.67 0.58 10.67 0.58 10.67 0.58 11.001.00 Staphylococcus aureus B 8.100.10 8.330.15 8.430.20 8.730.25 (-), no activity; L, leaf extract; B, bark extract; DW, distilled water Table 2: Inhibition zones of dichloromethane extract of A. crassna after 48 hours incubation Microorganism Plant Part Concentration of plant extract (mg/ml) 6 8 Inhibition zone (mm) 8.83 0.29 8.370.06 8.47 0.25 8.130.06 10.90 0.36 661 9.07 0.12 8.430.06 8.67 0.06 8.270.06 11.00 0.26

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Control DW

Gram-positive Pseudomonas aeruginosa Shigella flexneri Gram-negative Bacillus spizizenii Staphylococcus

L B L B L B L

8.57 0.40 8.230.06 8.00 0.00 8.100.10 9.83 0.64

9.33 0.58 8.600.00 9.10 0.10 8.370.06 11.33 0.61

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B 8.200.00 8.370.15 8.530.15 8.800.26 (-), no activityhy; L, leaf extract; B, bark extract; DW, distilled water The hexane extract of A. crassna leaf exhibited inhibition zones against two bacteria, namely Staphylococcus aureus and Shigela flexneri after the two incubation times (Table 3 and Table 4). The hexane extract of the bark, however, showed antibacterial activity towards Staphylococcus aureus alone. An increase in the inhibition zones occurred as the concentrations of extracts were increased. There were also no inhibition activity against Pseudomonas aeruginosa and Bacillus spizizenii after both incubation times. aureus Table 3: Inhibition zones of hexane extract of A. crassna after 24 hours incubation Microorganism Plant Part Concentration of plant extract (mg/ml) 6 8 Inhibition zone (mm) 9.00 0.00 9.00 0.00 Control DW

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Gram-positive Pseudomonas aeruginosa Shigella flexneri Gram-negative Bacillus spizizenii

L B L B

8.000.00 -

9.00 0.00 -

L B L 8.000.00 9.17 0.29 9.33 0.58 9.67 0.58 Staphylococcus aureus B 7.070.12 7.130.06 7.400.10 7.500.00 (-), no activity; L, leaf extract; B, bark extract; DW, distilled water Table 4: Inhibition zones of hexane extract of A. crassna after 48 hours incubation Microorganism Plant Part Concentration of plant extract (mg/ml) 6 8 Inhibition zone (mm) 9.07 0.12 9.30 0.17 -

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Control DW

Gram-positive Pseudomonas aeruginosa Shigella flexneri Gram-negative Bacillus spizizenii

L B L B

8.00 0.00 -

9.53 0.06 -

L B L 8.130.12 9.17 0.29 9.63 0.32 9.83 0.29 Staphylococcus aureus B 7.230.06 7.270.06 7.400.10 7.600.00 (-), no activity; L, leaf extract; B, bark extract; DW, distilled water The methanol extract of A. crassna leaf exhibited inhibition zones against two out of four bacteria tested at both incubation periods (Table 5 and Table 6). Interestingly, the methanol extract of bark showed inhibition against all four bacteria. The leaf methanol extract exhibited inhibition zones only against Staphylococcus aureus and Shigela flexneri. However, the inhibition zone for Pseudomonas aeruginosa show the lowest inhibition zones which 662

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measured only 6 mm compared to the other three bacteria that showed an average of 8 mm of antibacterial inhibition. Table 5: Inhibition zones of methanol extract of A. crassna after 24 hours incubation Microorganism Plant Part Concentration of plant extract (mg/ml) 6 8 Inhibition zone (mm) 6.700.00 9.00 0.00 8.330.06 6.830.06 9.00 0.00 8.400.00 Control DW

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Gram-positive Pseudomonas aeruginosa Shigella flexneri Gram-negative Bacillus spizizenii

L B L B

6.600.10 8.00 0.00 8.170.06

6.870.06 10.00 0.00 8.470.06

L B 8.030.06 8.170.06 8.230.06 8.400.10 L 10.000.00 10.00 0.00 10.33 0.58 11.00 0.00 Staphylococcus aureus B 8.000.00 8.170.06 8.330.06 8.470.06 (-), no activity; L, leaf extract; B, bark extract; DW, distilled water Table 6: Inhibition zones of methanol extract of A. crassna after 48 hours incubation Microorganism Plant Part Concentration of plant extract (mg/ml) 6 8 Inhibition zone (mm) 6.830.06 9.13 0.12 8.430.06 6.900.00 9.17 0.15 8.470.06

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Control DW

Gram-positive Pseudomonas aeruginosa Shigella flexneri Gram-negative Bacillus spizizenii

L B L B

6.770.06 8.03 0.06 8.300.00

7.030.06 10.00 0.00 8.570.06

L B 8.130.06 8.270.06 8.370.06 8.500.00 L 10.20 0.00 10.23 0.06 10.57 0.40 11.03 0.06 Staphylococcus aureus B 8.170.06 8.270.06 8.400.00 8.560.06 (-), no activity; L, leaf extract; B, bark extract; DW, distilled water The inhibition zones of the plant extracts against the four species of strain are categorized as highly inhibitory if the diameter of inhibition zone is (>20 mm), moderately inhibitory (10-19 mm), slightly inhibitory (1-9 mm) and non-inhibitory (<1 mm). The results obtained showed that the Staphylococcus aureus, a Gram-positive bacterium as the most susceptible strain towards all the six crude extracts, while Pseudomonas aeruginosa, a Gram-negative bacterium is the least susceptible strain. In all extracts, the inhibition zone became wider with the increase of the crude extract concentration. In addition, the incubation period affected the inhibition zone size whereby inhibition zones increased with the longer incubation time (48 hours).

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Conclusion All six crude dichloromethane, hexane and methanol extracts from the leaves and bark of Aquilaria crassna Pierre (Gaharu) showed promising antimicrobial inhibition toward the four bacteria strains. Staphylococcus aureus, showed the most susceptible strain towards all crude extracts, while Pseudomonas aeruginosa was found to be the least susceptible one. Acknowledgement The authors wished to thank UPSI for financial support via grant no. 04-16-0033-08 and MOSTI for providing the NSF scholarship. References [1] H.T. Loc and N.D.T. Luu: Conservation and use of Aquilaria crassna in Vietnam : A case study, central Forest company, Hanoi, Vietnam (2000) p. 155-157. [2] T. Yagura, N. Shibayama, M. Ito, F. Kiuchi and G. Honda: Four new 2-(2-Phenylethyl) chromone derivatives from withered wood of Aquilaria sinensis, Chem.Pharm. Bull 51 (5), 560-564. (2003) [3] T. Yagura, N. Shibayama, M. Ito, F. Kiuchi and G. Honda: Three novel diepoxy tetrahydrochromones from agarwood artificially produced by intention wounding, Tetrahedron Letters, Vol 46, Issue 25, 4395-4398. (2005) [4] M. Zhou, H. Wang, S. Jiba, J. Kou and B. Yu: Antinociceptive and anti-inflammatory activities of Aquilaria Senensis (Lour) Gilg. Leaves Extract, Journal of Ethnopharmacology, 117, 345-350. (2008) [5] M. Dash, J.K. Patra, P.P. Panda:. Phytochemical and antimicrobial screening of extracts of Aquilaria agallocha Roxb, African Journal of Biotechnology,7 (20), 3531-3534. (2008)

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