lsp99 - s.s. syed abdul azziz.pdf

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UMTAS 2011Empowering Science, Technology and Innovation Towards a Better Tomorrow

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LSP99

Biological Activities of Leaf and Bark from Aquilaria crassna Pierre(Gaharu)

H. Alimon 1, N. Mohd Arriffin 2, S.S. Syed Abdul Azziz 2, R. Ibrahim 2, F. MohdJaafar 3 and M.A. Mohd Sukari 4

1Department of Biology, 2Department of Chemistry, Faculty of Science and Mathematics, UniversitiPendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia

3Department of Chemistry, Faculty of Applied Sciences, Universiti Teknologi Mara, 40450 ShahAlam , Selangor, Malaysia

4Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor,Malaysia

Email: [email protected]

Abstract Crude extracts of leaves and bark of Aquilaria crassna Pierre (Gaharu) using three differentsolvents: hexane, dichloromethane and methanol, had been screened for antimicrobialactivities toward four bacteria; Pseudomonas aeruginosa , Bacillus spizizenii , Staphylococcusaureus and Shigela flexneri . The results showed that Staphylococcus aureus was the mostsusceptible strain to all six crude extracts, in which the highest inhibition was exhibited bythe methanol extract of the leaf. The least susceptible strain was Pseudomonas aeruginosa ofwhich only the bark methanol extract showed the inhibition zone.

Key words: Aquilaria crassna, Thymelaeceae, antimicrobial

AbstrakEkstrak daun dan batang Aquilaria crassna Pierre (Gaharu) menggunakan tiga jenis pelarut

berbeza; heksana, diklorometana dan metanol, telah disaring untuk aktiviti antimikrobterhadap empat bakteria; Pseudomonas aeruginosa , Bacillus spizizenii , Staphylococcusaureus dan Shigela flexneri . Keputusan menunjukkan Staphylococcus aureus adalah strainyang paling mudah direncat oleh kesemua enam ekstrak mentah, dengan perencatan tertinggiditunjukkan oleh ekstrak metanol daun. Manakala, strain yang paling kurang direncat adalahPseudomonas aeruginosa, iaitu hanya ekstrak metanol batang menunjukkan zon perencatan .

Kata kunci: Aquilaria crassna, Thymelaeceae, antimikrobial


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IntroductionGaharu or agarwood (aloeswood, eaglewood) is a resinous, fragrant and highly valuableheartwood of Aquilaria species from the Thymelaeceae family. There are altogether 16

species of Aquilaria . The different species was found in different country . However, the bestknown 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 15-20m 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 inimportant religious ceremonies, rituals and meditation. Gaharu is widely used in traditionalmedicine as sedative, analgesic and digestive [2,3]. The gaharu oil is highly demanded for itsuse in perfumes and toiletry products such as shampoo and soap. Recently phytochemical and

pharmacological studies on gaharu have increased. However, previous scientific work hadonly 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 treatmentof trauma-related diseases such as fractures and bruises. Unfortunately relatively little isknown 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. Thezone of clearance around each well was measured after the incubation period. The resultsshowed that there were inhibition zones around the well and confirmed the antimicrobialactivity 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 highestinhibition zone with 19 mm against B. subtilis , the methanol extracts of bark did not showany 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 thatalso act as antimicrobial compounds.

Materials and MethodsThe leaves and barks of Aquilaria crassna was collected from Kajang, Selangor, Malaysia onJuly 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 andevaporated under reduced pressure to give their crude extracts. A total of six plant extractswere 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. Themixture 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 extractsstock 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 (ATCC10145) and Shigella flexneri (ATCC 12022). The bacteria were purchased from Choice CareSdn. Bhd. The test organisms were purified and maintained on slant agar kept at 4 o C untilfurther 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 about15 minute at 121 oC. The sterilized melted agar was poured into sterile Petri dishes in thelaminar flow for the agar plate preparation.

Nutrient Broth. 13 g nutrient broth was suspended with 1 liter of distilled water. The mediadissolved by fast cooking in the microwave. 5 ml of melted broth were pipetted into test tubeand sterilized by autoclaving for about 15 minutes at 121 oC.

Screening for Antibacterial Activity. The antibacterial activity of the extracts on themicroorganisms 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 inhibitionin growth of bacteria by different concentrations of the plant extract on paper disc. Thevolumes of extract used were 20 l, 30 l, 40 l and 50 l from the 200 mg/ml of each plantextract 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 wasaseptically introduced and spread on the surface of sterile nutrient agar using a sterile cottonswab. 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 37 0C for 24-48 hours. All experiments were performed in triplicateand each experiment was reproduced a minimum of three times and all these procedures werecarried out aseptically.

Determination of Antibacterial Properties. Reading of the inhibition zones was done at 24 and48 hour-intervals. The antibacterial activity was interpreted from the size of the diameter ofzone inhibition measured to the nearest mm as it is observed from the clear zone surroundingthe disc. The zone of inhibition is measured from the edge of the disc to the edge of thegrowth. It is measured on the undersurface of the plate without opening the lid.


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Results and DiscussionSix 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 similarinhibition zones against three out of four bacteria tested for both incubation times (Table 1and Table 2); Bacillus spizizenii , Staphylococcus aureus and Shigela flexneri. An increase inthe inhibition zones occurred as the concentration of the extracts increased. However, therewas not much difference in the size of the inhibition zones to the incubation times for allthree bacteria. Nevertheless, the dichloromethane extracts showed highest inhibition growthagainst Staphylococcus aureus whereas no inhibition activity was shown againstPseudomonas aeruginosa at the two incubation times.

Table 1: Inhibition zones of dichloromethane extract of A. crassna after 24 hours incubation

Microorganism PlantPart

Concentration of plant extract (mg/ml) ControlDW4 6 8 10

Inhibition zone (mm)Gram-positivePseudomonas

aeruginosaL - - - - -B - - - -

Shigella flexneri L 8.33 0.58 8.67 0.58 9.00 0.00 9.33 0.58 -

B 8.100.10 8.270.06 8.330.06 8.500.00Gram-negative

Bacillus spizizenii 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

Staphylococcusaureus

L 9.67 0.58 10.67 0.58 10.67 0.58 11.001.00 -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 PlantPart Concentration of plant extract (mg/ml) ControlDW4 6 8 10Inhibition zone (mm)

Gram-positivePseudomonas


Shigella flexneri L 8.57 0.40 8.83 0.29 9.07 0.12 9.33 0.58 -B 8.230.06 8.370.06 8.430.06 8.600.00

Gram-negative

Bacillus spizizenii L 8.00 0.00 8.47 0.25 8.67 0.06 9.10 0.10 -

B 8.100.10 8.130.06 8.270.06 8.370.06Staphylococcus L 9.83 0.64 10.90 0.36 11.00 0.26 11.33 0.61 -


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aureus 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, namelyStaphylococcus aureus and Shigela flexneri after the two incubation times (Table 3 and Table4). The hexane extract of the bark, however, showed antibacterial activity towardsStaphylococcus aureus alone. An increase in the inhibition zones occurred as theconcentrations of extracts were increased. There were also no inhibition activity againstPseudomonas aeruginosa and Bacillus spizizenii after both incubation times.

Table 3: Inhibition zones of hexane extract of A. crassna after 24 hours incubation





Shigella flexneri L 8.000.00 9.00 0.00 9.00 0.00 9.00 0.00 -B - - - -

Gram-negative

Bacillus spizizenii L - - - - -B - - - -


L 8.000.00 9.17 0.29 9.33 0.58 9.67 0.58 -B 7.070.12 7.130.06 7.400.10 7.500.00


Table 4: Inhibition zones of hexane extract of A. crassna after 48 hours incubation





Shigella flexneri L 8.00 0.00 9.07 0.12 9.30 0.17 9.53 0.06 -B - - - -

Gram-negative Bacillus spizizenii L - - - - -

B - - - -Staphylococcus

aureusL 8.130.12 9.17 0.29 9.63 0.32 9.83 0.29 -B 7.230.06 7.270.06 7.400.10 7.600.00


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 methanolextract 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


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measured only 6 mm compared to the other three bacteria that showed an average of 8 mm ofantibacterial inhibition.

Table 5: Inhibition zones of methanol extract of A. crassna after 24 hours incubation

Microorganism Plant

Part

Concentration of plant extract (mg/ml) Control

DW4 6 8 10Inhibition zone (mm)

Gram-positivePseudomonas

aeruginosaL - - - - -B 6.600.10 6.700.00 6.830.06 6.870.06

Shigella flexneri L 8.00 0.00 9.00 0.00 9.00 0.00 10.00 0.00 -B 8.170.06 8.330.06 8.400.00 8.470.06

Gram-negative

Bacillus spizizenii 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 -B 8.000.00 8.170.06 8.330.06 8.470.06


Table 6: Inhibition zones of methanol extract of A. crassna after 48 hours incubation



Inhibition zone (mm)Gram-positive

Pseudomonasaeruginosa L - - - - -B 6.770.06 6.830.06 6.900.00 7.030.06Shigella flexneri L 8.03 0.06 9.13 0.12 9.17 0.15 10.00 0.00 -

B 8.300.00 8.430.06 8.470.06 8.570.06Gram-negative

Bacillus spizizenii 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 -B 8.170.06 8.270.06 8.400.00 8.560.06


The inhibition zones of the plant extracts against the four species of strain are categorized ashighly inhibitory if the diameter of inhibition zone is (>20 mm), moderately inhibitory (10-19mm), slightly inhibitory (1-9 mm) and non-inhibitory (


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ConclusionAll 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 allcrude extracts, while Pseudomonas aeruginosa was found to be the least susceptible one.

AcknowledgementThe authors wished to thank UPSI for financial support via grant no. 04-16-0033-08 andMOSTI 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. Bull51 (5), 560-564. (2003)

[3] T. Yagura, N. Shibayama, M. Ito, F. Kiuchi and G. Honda: Three novel diepoxytetrahydrochromones 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-inflammatoryactivities of Aquilaria Senensis (Lour) Gilg. Leaves Extract, Journal ofEthnopharmacology, 117, 345-350. (2008)

[5] M. Dash, J.K. Patra, P.P. Panda:. Phytochemical and antimicrobial screening of extractsof Aquilaria agallocha Roxb , African Journal of Biotechnology,7 (20), 3531-3534.(2008)

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