Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE)

78

Research Article

Characterization of Methanolic Extracts of Agarwood Leaves

Khalil, A. S.1; Rahim, A. A.2, Taha, K. K.*3, Abdallah, K. B. 4

1

Department of Chemistry and Industrial Chemistry, College of Applied and Industrial Sciences, University of Bahri, Sudan 2 School of Chemical Sciences, Universiti Sains Malaysia (USM), Penang- Malaysia
4

*3Department of Chemistry, College of Science, Al-Imam Mohammad Ibn Saud Islamic University, P.O. Box : 5701, Riyadh, KSA Department of Chemical Engineering, College of Engineering, Karary University, Sudan (Received: June 14, 2013; Accepted: August 21, 2013)

Abstract Tropical Agarwood (Aquilaria) is in danger of extinction in the wild due to illegal logging. Its resin (Gaharu) is used for the production of highly valued incense throughout Asia. The methanolic crude extracts of Agarwood leaves before and after inoculation process extracted by using maceration into methanol as solvent were screened for Phytoconstituents, the extracts revealed the presence of alkaloids, tannins, saponins, flavonoids and terpenoids. Gas chromatography-mass spectrometry (GC-MS) analysis of the plant extracts led to the identification of 14 components from the two types of Agarwood leaves extracts. It is interesting to see here that all the identified compounds from agarwood leaves Methanolic extracts contained oxygen and/or -electrons in their molecules. Hexadecanoic acid was found to be one of the major compounds in Agarwood leaves methanolic extracts. Infrared spectroscopic analysis of the extracts of Agarwood revealed the presence of O-H, C=O, C-H, C-N, C-O, -CH2 and CH3 bond stretching. The present study has proved the usefulness of agarwood tree for medicinal and anti corrosive purposes. The presence of phytochemicals indicates its potential as a source of useful drugs. Index Terms Agarwood, Inoculation, Methanolic extract, Phytoconstituents

promising potential antidiabetic agent [4]. The extracts of this plant, which contains numerous naturally environmental organic compounds, may be utilized as eco-friendly natural corrosion inhibitors for metals or alloys in aggressive medias. There is an intensive effort underway to develop new plant origin corrosion inhibitors for metal subjected to various environmental conditions. These efforts have been motivated by the desire to replace toxic inhibitors used for mitigation of corrosion of various metals and alloys in aqueous solutions. Plants represent a class of interesting source of organic compounds currently being explored for use in metal corrosion protection in most systems, as possible replacement of toxic synthetic inhibitors. Aquilaria Malaccensis (Agarwood) is one of 15 tree species in the Indomalesian genus Aquilaria, family Thymelaeaceae, [5] and class Magnoliopsida. It is a large evergreen tree growing over 15-30 m tall and 1.5- 2.5 m in diameter, and has white flowers [6]. The Leaves are 5-11 cm long and 2-4 cm broad. Aquilaria species have adapted to live in various habitats, including those that are rocky, sandy or calcareous, welldrained slopes and ridges and land near swamps. They typically grow between altitudes of 0-850 m, in locations with average daily temperatures of 20-22 C [7]. Aquilaria Malaccensis (Agarwood) is widely distributed in south and south-east Asia. There are differing accounts of the countries in which it occurs. According to Oldfield, S., et al. (1998) [8], A. Malaccensis is found in 10 countries: Bangladesh, Bhutan, India, Indonesia, Iran, Malaysia, Myanmar, Philippines, Singapore and Thailand. Agarwood, eaglewood, gaharu, aloeswood, oud, oudh, kanankoh, kyara, jinkoh, chen xiang, tomention and kalamabak- these are just a few of the names for this tree. Agarwood is the most valuable wood in the world with higher prices and demands nowadays. The widely uses of agarwood in meditation field, essential oil production and etc makes agarwood one of the precious things on earth. There is no detail systematic documentation of presence and type of phytochemicals in agarwood leaves. Hence the present study aimed at an evaluation of the presence of

I. INTRODUCTION

nowledge of the chemical constituents of plant is helpful in the discovery of therapeutic agent as well as new sources of economic materials like oil and gums. The most important bioactive constituents of these plants are alkaloids, tannins, flavonoids and phenolic compounds [1]. Studies revealed that agarwood has remarkable anticancer activity [2]. The Benzene extracts of the plant have central nervous system antidepression activities [3]. Rise in demand for agarwood resulted in irrational cutting of the tree trunk for extraction of the chemicals. This has resulted in the tree becoming endangered. Agarwood leaf is a
Corresponding author E mail: kamaltha1012@yahoo.com

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) different phytochemicals along with GC-MS and FTIR investigations of methanol soluble crude extracts obtained from before and after inoculation leaves of Aquilaria Malaccensis (Agarwood). II. MATERIALS AND METHODS Collection of the Plant Materials Two types of fresh leaves of agarwood were collected from (Main land-Penang, Malaysia). One was before the inoculation process (MOB) and the other type was after the inoculation process (MOA). Both new and old growth leaves were avoided, damaged and diseased ones were excluded, only recently matured leaves were taken. Preparation of the Plant Materials The leaves were dried in the shade for 7 days at room temperature (28 2C) and ground to a fine powder using Grinder IKA-WERKE, IKA MF10 Machine and sieved through a 0.25 m mesh. The powder samples were kept at room temperature in a covered glass containers to protect them from humidity and light prior to extraction. Extraction of Agarwood Leaves Extracts 250g dried powder leaves of each A. Malaccensis (Agarwood) type (before and after inoculation) were exhaustively extracted by macerated in 1.5 L methanol solvent for 2 days at room temperature (282C). The solvent-containing extract was then decanted and filtered by vacuum filtration (GAST, DOA-P504-BN, USA). The extraction of the ground leaves were further repeated (twice) with methanol (1 L each time). The filtrate from each extraction was combined and the excess solvent was evaporated under reduced pressure at 40C using a rotary evaporator (Heidolph-instruments, Rotavapor, Germany) to give concentrated crude methanolic extracts, dried in oven at 50C to give dark green extracts. The weights of all the extracts were measured after solvent evaporation and then kept into a glass container prior to use. The above mentioned processes were done for each type of leaves separately. Preliminary Phytochemical Analysis of the Agarwood Methanolic Crude Extracts Extracts were tested for the presence of active principles such as alkaloids, flavonoids, saponins, steroids, terpenoids and tannins. Following standard procedures were used Alkaloids Determination The presence of alkaloid was determined using the Mayer and Dragendorff tests as described by [9 and 10]. 0.2 g of each extract of the sample were put into a conical flask, 20 ml of dilute sulphuric acid in methanol were added and then heated in water bath to boil for 5 min. The mixture was filtered (vacuum pump) and the filtrates were separated and treated with 2 drops of Mayer`s (Potassium mercuric iodide solution) and Dragendorffs (Potassium bismuth iodide solution) reagents in test-tubes. Development of creamy and an orange color respectively indicated positive result.

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Flavonoids Determination i. Ammonium Test: The presence of flavonoids in the samples was determined using the Harbone and Sofowora methods [9 and 11]. 10 ml of ethyl acetate was added to 0.2 g of the extract and heated in a water bath for 5 min. The mixture was cooled filtered and the filtrates used for the test. About 4 ml of filtrate was shaken with 1 ml of dilute ammonia solution. The layers were allowed to separate and the yellow color in the ammonical layer (bottom layer) indicates the presence of flavonoids. ii. Shinoda Test: Small pieces of Magnesium ribbon followed by few drops of concentrated hydrochloric acid were added to a small amount of methanolic extract of the plant material. Immediate development of a pink scarlet or crimson red color was taken as an indication of the presence of flavonoids [9 and 10]. Saponins Determination The froth test and emulsion test as described by Harbone 2001 were used to determine the presence of saponins. 20 ml of water was added to 0.25 g of the extract in 100 ml beaker and boiled, filtered and then the filtrates used for the tests: i. Froth Test: 5 ml of the filtrate was diluted with 20 ml of water and shaken vigorously. A stable froth (foam) up on standing indicates the presence of saponins [11] ii. Emulsion Test: 2 drops of olive oil was added to the frothing solution and shaken vigorously the formation of emulsion indicates the presences of saponins. Steroids / Terpenoids Determination i. Liebermann-Burchardt test: 1 ml of methanolic extract of each sample is boiled with 23 ml of acetic anhydride, and then cooled; 1 to 2 drops of concentrated sulfuric acid were added slowly through the wall of the tube. Dark green coloration of the solution indicates the presence of Steroids and dark pink or red coloration in the interface indicate the presence of Terpenoids. ii. Salkowski test: 1 ml of methanolic extract of each sample is boiled with 2 ml chloroform, cooled, 1 to 2 drops of concentrated sulfuric acid were added slowly through the wall of the tube. Shake well and allow standing for some time, red color appears at the lower layer indicates the presence of Steroids and formation of yellow colored lower layer indicates the presence of triterpenoids. Tannins Determination i. Ferric chloride test: About 0.5 g of the methanolic extract was dissolved in 5 to 10 ml of distilled water and filtered. A few drops of a 10% ferric chloride solution were added to the filtrate. A greenish black colour or a precipitate was taken as an indication of the presence of tannins [9 and 10]. ii. Alkaline reagent test: Test solution with sodium hydroxide solution gives yellow to red precipitate within short time.

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) Preliminary Phytochemical Analysis of the Agarwood Methanolic Crude Extracts The qualitative screening of phytochemical compounds in Agarwod methanolic extracts revealed the presence of alkaloids, saponins and phenolic compounds (flavonoids, terpenoids, and tannins). The presence of phytochemicals indicates its potential as a source of useful corrosion inhibitors. Factors affecting capture of active phytochemicals are the plant part being extracted, the types of solvents, the extraction period and extraction conditions [12 and 13]. For example, antioxidant in plant material may be water soluble, fat soluble, insoluble or bound to cell walls, and react at different rates. Therefore, methanol was chosen in this study as the organic solvent for its wide solubility properties for low molecular weight and moderately polar substances, including phenolic compounds. However, despite these challenges, qualitative screening of native plants is useful for revealing activity that may lead to the development of new products for use as anticorrosion. The phytochemical compounds of plants have potentially significant application in human health care as well as in corrosion field. The presence of each secondary metabolite in Agarwood leaves extracts gives a justification for the traditional use of the plant in treating various health problems. For instance, detection of alkaloids in Agarwood leaves extracts was of great importance, since significant quantities of alkaloids could be used as antimalaria, analgeics, antispasmodic, bactericidal and stimulants, which are all pharmaceutical properties of the plant. Presence of alkaloids in Agarwood leaves extracts, justifies the use of the plant to treat toothache, colic, severe headache, rheumatism and pains during pregnancy. On the other hand, flavonoids, which are a group of polyphenolic compounds, have been reported to have antiinflammatory action, free radical scavenging and inhibition of hydrolytic and oxidative enzymes [14], anti-allergic, anticancer, anti-inflammatory and anti-viral [15]. Moreover, the presence of glycosides moieties like saponins, anthraquinones, cardiac glycosides and flavonoids could inhibit tumor growth and protect against gastrointestinal infections [16 and 17]. Corrosion inhibition performance of three flavonoids, apigenin, luteolin-3-methyl ether and quercetin-3,3dimethylether on copper dissolution in 2.0 M HNO3 was investigated by [18]. 92% Inhibition was observed in some of these flavonoids. The inhibitive action of commercial green tea extracts flavonoids monomers of tea catechins on mild steel (MS) in a 1.0 M hydrochloric acid solution was investigated by [19], 84% Inhibition was observed in one of the green tea extracts. Herbs that have tannins as their components are astringent in nature, i.e., fasten the healing of wounds and inflamed mucous membranes and could be used for treating intestinal disorders such as diarrhea and dysentery exhibiting antibacterial activity [20]. Furthermore, polyphenols are known to be responsible for antioxidant and antimicrobial activities [20 and 21]. Recent

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Characterization of Agarwood Methanolic Extracts by Fourier Transforms Infrared (FTIR) All extracts were characterized by FTIR spectroscopy (Perkin Elmer 2000 Model) for identification of the active functional groups. The FTIR study was carried out by using the Perkin Elmer System 2000 FTIR instrument. First, the transparent Pellets (thin disc) were formed by mixing 5mg of the sample with 100 mg of potassium bromide (KBr) (1:20) using a mould and press, and compressed under a pressure of 7 ton. The investigation was performed within the wavelength ranging from 4000 to 400 cm-1 and the spectrum takes about three minutes to be recorded. The acquisition of the spectra and peaks assignment was performed using FTIR software Spectrum Version 3.02.01 (Perkin Elmer, Inc., Waltham, MA). Gas chromatography-Mass Spectrometry (GC-MS) Analysis Gas ChromatographyMass Spectrometry (GCMS) analysis was carried out for the all methanolic extracts using Perkin Elmer, Clarus 600 gas chromatograph combined to Perkin Elmer, Clarus 600 T Mass Spectrometer. The methanolic extracts were redissolved in methanol and filtered using 0.45 m pore size whatman polyethersulfone membrane filter. The analysis was performed according to the method described by Soetardjo et al, 2007. Semi-standard non polar fused silica DB-1 capillary column (30 m long * 320 m inter nal diameter) served as stationary phase. The column temperature was programmed to 50C for 6 min, with 10C increase per min to 250C, which was maintained for 30 min. The injector and detector temperatures were both maintained at 250C. Helium was used as a carrier gas at a flow rate of 1 ml/min; splitting ratio was: 10:1. The Mass spectral ionization temperature was set at 250 C. The mass spectrometer was operated in the electron impact ionization mode at a voltage of 70 eV. Mass spectra were taken over the m/z range 28400 atomic mass unit (amu). The compounds of each plant extracts were identified by computer searches in commercial libraries of WILEY and NIST (National Institute of Standards and Technology). III. RESULTS AND DISCUSSION The percentage yield of the extracts was based on the weight of dried and ground plant materials. Beside the single solvent extraction, four solvents extraction was used in another study, namely n-hexane (C6H14), dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and methanol (CH3OH). The percentage yield of the methanolic extracts of Agarwood leaves (MOB and MOA) were shown in Table 1. Table 1 The physical properties of dry crude methanolic extracts of the Agarwood leaves
Extract Physical characteristics Percentage yield (%) Color Odor Consistency MOB extract 52.12 Dark green Leafy smell but sweet odor Waxy thick MOA extract 39.6 Dark green Leafy smell but sweet odor Waxy thick

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) epidemiological studies have strongly suggested that consumption of certain plant materials rich in phenolic compounds may reduce the risk of chronic diseases related to oxidative stress on account of their antioxidant activity and thus promote general health benefits. Therefore, the significant antibacterial, anticandida and antioxidant activities of Agarwood plant could be related to the presence of phenolic compounds. Catechin, epigallocatechin gallate, epicatechingallate, epigallocatechin and epicatechin that constitute Mangrove tannins were investigated in an aerated HCl, the monomers were found to be mainly cathodic inhibitors and the inhibition efficiency was dependent on concentration. Saponin has the property of precipitating and coagulating red blood cells. Some of the characteristics of saponins include formation of foams in aqueous solutions, hemolytic activity, cholesterol binding properties and bitterness [22]. These properties bestow high medicinal activities on the extracts. It has also been shown that saponins are active antifungal agents [23]. This therefore supports the earlier finding that extracts of the plant used in the present work may be useful in the chemotherapy of mycotic infections. Presence of tannins suggests the ability of this plant to play a major role as antidiarrhoec and antihaemorrhagic agent [24]. The presence of mimosa tannin as a low carbon steel corrosion inhibitor in sulphuric acid media was tested [25] in concentrations from 105 to 101 mol/l, at the temperature of 298 K in the solutions of pH 1, 2 and 3. The inhibitor effectiveness increases with increase in concentration. Beside the anti corrosion activity of methanol soluble leaf extracts, the present study indicates the usefulness of agarwood tree for medicinal purposes. The presence of phytochemicals indicates its potential source of useful drugs (further study is recommended).

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Table 2 Summary of the phytochemical screening of crude Methanolic Extracts of Agarwood leaves (before and after inoculation) Extract Test Alkaloids test (Mayers Reagent) Alkaloids test (Dragndorffs Reagent) Triterpenoid / Steroid (Salkowski-Liberman) Flavonoids test (Harbone and Sofowora methods) -Ammonium test Saponin test (Froth test) Saponin test-Emulsion test Before Inoculation (MOB) Observation Result After Inoculation (MOA) Observation Result

Creamy ppt.

Dense Creamy ppt.

Orange red ppt. Reddish brown ring in the interface Yellow color obtained in the ammonical layer (lower layer) Stable persistent froth(thick layer) Formation of emulsion

+ +T

Reddish brown ppt Reddish brown ring in the interface Clear yellow color obtained in the ammonical layer (lower layer) Stable persistent froth(thin layer) Formation of emulsion Greenish black solution (ppt.)

+ +T

+ + +

+ + +

Dark green solution Tannin test (Harbone, (ppt.) Braemers methods) +: Positive result (Presence of the phytochemical) +T: Positive result for triterpenoid

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) Table 3 Summary of the phytochemical screening tests on Methanolic crude extracts of Agarwood leaves (Before and After inoculation)
Alkaloids test (Mayers Reagent) Alkaloids test (Dragndorffs Reagent) Flavonoids test (Harbone and Sofowora methods) Triterpenoid / Steroid (SalkowskiLiberman) Saponin test (Froth test) Saponin test (Emulsion test) Tannin test (Harbone, Braemers methods)

82

Crude Extract

MOB + + + +T ++ ++ +

MOA +++ ++ ++ ++T + + ++

+: Positive result: + Low, ++ Medium, +++: High +T: Positive result for Triterpenoid Medium, ++T: High Characterization by Fourier Transform Infrared (FTIR) analysis The infrared spectra of all the extracts were taken and compared. The spectrums did not shows significant changes of peaks for Agarwood leaves before and after inoculation process. Figures 1a, 1b show the IR spectrum of MOB, MOA respectively. Figure 2 shows the infrared spectra for all the methanolic extracts of Agarwood leaves before and after inoculation process with its adsorption characteristic region for OH, C=C and C-O groups (in horizontal line). The spectrums did not show significant changes of peaks for Agarwood leaves before and after inoculation process. FTIR spectrum of methanol only extract before inoculation (MOB) shows the main characteristic absorption broad band appeared at 3388 cm-1 assigned to the presence of (OH/NH) group; the presence of the methyl (CH3) and methylene ( CH2) aliphatic saturated (CH) sharp asymmetric and symmetric stretching band which are observed at 2924 cm-1 and 2852 cm-1, respectively. The (C=O) stretching band is observed at 1709 cm-1; it is due to the presence of carboxylic acid/ketones groups. The band at 1621 cm-1 shows the presence of aromatic ring (C=C).The peak at 1455 cm-1 is due to aromatic (CC) stretching band. The peak at 1280 cm-1 is due to aromatic (COC) band. The (CN) medium stretching band and/or PH phosphine band were observed at 1076 cm-1. Absorption band appeared at 827 cm-1 assigned to (SOR) esters. Furthermore, the adsorption band around ~ 1200 900 cm-1 and~ 1100700 cm-1 may represents to the 1,2disubstituted and 1,3-disubstituted benzene ring [26], (Figure 1a).

FTIR spectrum of methanol only extract after inoculation (MOA) shows (Figure 1b) the main characteristic absorption broad band appeared at 3384 cm-1 assigned to the presence of (OH/NH) group; the presence of the methyl (CH3) and methylene (CH2) aliphatic saturated (CH) sharp asymmetric and symmetric stretching ba nd which are observed at 2923 cm-1 and 2851 cm-1, respectively. The (C=O) stretching band is observed at 1708 cm-1; it is due to the presence of carboxylic acid group. The band at 1618 cm-1 shows the presence of aromatic ring (C=C).The peak at 1463 cm-1 is due to aromatic (CC) stretching band. The peak at 1290 cm-1 is due to aromatic (COC) band. The (CN) medium stretching band and/or PH phosphine band were observed at 1078 cm-1. The aromatic bending band occurs at the range of 827-787 cm-1. Absorption band appeared at 827 cm-1 assigned to (SOR) esters. Furthermore, the adsorption band around ~ 1200900 cm-1 and~ 1100700 cm-1 may represents to the 1,2disubstituted and 1,3-disubstituted benzene ring [26], (Figure 1b).

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE)
66.4 65

83

60

55
827

50 %T 45
1167

787 611

40
2852

1377 1455

1280

35
1621 3388 2924

1076

29.4 4000.0 3000 2000 cm-1 1500 1000 500 370.0

Fig. 1a: FTIR Spectrum for Agarwood methanolic extract before inoculation (MOB)

76.4

74

72

70

%T 68 66
1730 1708 2851 1290 14631380 2923 1618 1078 3384

610

64

62

60.1 4000.0 3000 2000 cm-1 1500 1000 400.0

Fig. 1b: FTIR Spectrum for Agarwood methanolic extract after inoculation (MOA) The presence of atheroline alkaloids was supported by using FTIR studies. It showed the presence of aromatic rings (C=C stretching bands) between 1621 and 1624 cm-1 and the O-Me group appeared from 1383 to 1075 cm-1. Moreover, a broad band of the FTIR spectrum at around 3400 cm-1 showed the presence of N-H groups. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis Fourteen different compounds were identified by GC-MS analysis of the Agarwood leaves methanolic extracts. Hexadecanoic acid was found to be one of the major compounds in Agarwood leaves methanolic extracts. It was eluted at the same retention time with large abundance. Hexadecanoic acid was one of the most common saturated fatty acids found in animals and plants, and was reported to have potential antibacterial and anti fungal activity. In one study by Saidana, [27], Hexadecanoic acid had been reported to exhibit antibacterial activity against Gram-positive and Gram-negative bacteria comparable to ampicillin and in the case of Streptococci, greater than that of ampicillin. In another study by [28], Hexadecanoic acid was considered as the major antibacterial compound in the ethyl acetate root extract of Pentanisia prunelloides. Thus, the presence of Hexadecanoic acid could justify the use of Agarwood leaves methanolic extracts as antibacterial and anti fungal as well as anti corrosion especially in that type of corrosion caused by

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) bacteria in underground pipes. So further study of Hexadecanoic testing as anti corrosion in microbiological corrosion is recommended. The gas chromatograms of the methanolic extracts of A. Malaccensis leaves are shown in Figures 3a and 3b while the compounds and their corresponding mass spectra are shown in Table 5 and 6, the retention times (Rt) were reported in minutes. The major compounds identified in MOB extract were 1, 4, 7, 10, 13-Pentaoxacyclopentadecane (Rt 2.52, 13.9%); 1, 4, 7, 10, 13, 16-Hexaaoxacyclooctadecane (Rt 4.44, 8.17%), Acetic acid (Rt 6.2, 6.87%) and Hexadecanoic acid (Rt 19.82, 4.15%); in MOA were 1, 4, 7, 10, 13Pentaoxacyclopentadecane (Rt 2.52, 20.6%), Hexaethylene glycol monododecyl ether (Rt 3.8, 21.56%) and 2, 6, 10, 14, 18, 22- tetracosahexaen, 2, 6, 10, 15, 19, 23-hexamethyl- (Rt 21.17, 4.43%);

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Table4 The summary of characteristic peaks bands on FTIR spectra for all the methanolic extracts Wavenumber (cm-1) Functional groups (bands) MOB extract MOA extract 3388 3384 OH (stretch) (b),(str) (b),(str) CH alkane stretching 2924(str) 2923(str) C=O stretching 1709(m) 1708(str) C=C aromatic 1621 1618 stretching (b), (str) (b), (str) Asymmetric 1511(str) 1509(str) RCOO- stretching CC stretching 1455(str) 1463(str) Aromatic hydroxyl 1377(str) 1380(str) deformation COC cyclic ether 1280(str) 1290(str) Aromatic CO 1167(m) 1166(str) stretching Aliphatic CO/CN/P1076(str) 1078(str) H stretching (Note; (b): broad, (s): strong, (m): medium, (w): weak)
C=C OH C-O

Fig2: Fourier transform infrared spectrum. The black spectrum) methanolic extract corrosion inhibitor (MOB) (uninoculated leaves); and the blue spectrum) methanolic extract corrosion inhibitor (MOA) (inoculated leaves);

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) Gas chromatography-mass spectrometry (GC-MS) analysis of plant extracts led to the identification of 14 components from the two types of Agarwood leaves extracts (Table 7). It is interesting to see here that all identified compounds from plant extracts contained oxygen and/or -electrons in their molecules. Moreover, from the previous studies on the phytochemical constituents of the plant extracts it was established that the plant extracts used in this study also contain a mixture of organic compounds containing O, N or electrons in their molecules. Hence, the corrosion inhibition of carbon steel through these studied plants may be attributed to the adsorption of the phytochemicals containing O, N or electrons in their molecules as these atoms are regarded as centers of adsorption onto the metal surface. However, the highly complex chemical compositions of the plant extracts make it rather quite difficult to assign the inhibitive effect to a particular compound present in plants extracts. Having confirmed the corrosion inhibition effectiveness of these plants extracts, further detailed investigation for each plant extracts through inhibitive assay guided isolation using surface analytical techniques will enable the characterization of the active compounds in the adsorbed layer and assist in identifying the most active phytochemicals.

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Fig. 3a: Gas chromatogram of the Methanolic extracts of Agarwood leaves before inoculation (MOB)

Fig. 3b: Gas chromatogram of the Methanolic extracts of Agarwood leaves after inoculation (MOA)

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) Table5: Results of the GC-MS analysis of Agarwood leaf extract (MOB)
Percent No. Rt(min) of total (%) Compound Structure

86

19.816

4.153

n-Hexadecanoic acid, methyl ester

9,12,152 23.207 3.382 octadecatrienoic acid,(Z,Z,Z)-

2,3,Dihydro-3,53 14.632 Dihydroxy-6methyl-4H-pyran4-one

Table6: Results of the GC-MS analysis of Agarwood leaf extract (MOA)

Percent No. Rt(min) of total (%) Compound Structure

n-Hexadecanoic 1 19.816 3.876 acid, methyl ester

17.672

0.629

Phytol

14.992

0.921

1,2,3propanetriol

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE) Table 7: Chemical components identified in Agarwood leaves methanolic extracts by GC-MS. Molecular No. Component MOB MOA Formula C5H10O4 C12H16O6 C16H32O2 C3H8O3 C12H24O6 C3H6O3 + -

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1 2 3 4 5 6

1,2,3-propanetriol, monoacetate Phenyl--D-glucoside n-hexadecanoic acid Glycerine Squalene 2-propanone,1,3 dihydroxy 3,7,11,15-Tetramethyl-2-Hexadecen-

+ + + +

+ + + +

7 1-ol (Phytol) 8 Dodecyl acrylate 2,3-Dihydro-3,5-Dihydroxy-6-methyl9 4H-pyran-4-one 6-ethyl-5-hydroxy-2,3n,710 trimethoxynaphthoquinone

C20H40O

C15H28O2

+ +

+ +

C6H8O4

C15H16O6

11

9,12,15-Octadecatrienoic acid, (z,z,z)-

C18H30O2

12

Squalene

C30H50

13

Octaethylene glycol monodecyl ether

C28H58O9

14

1-Tetradecanol (): absence

C14H30O

(+): present

ACKNOWLEDGEMENT Greatest thanks to Dr. Afidah Abdul Rahim- Universiti Sains Malaysia (USM), School of Chemical Sciences, PenangMalaysia for providing the leaves of Aquilaria Malaccensis

and for giving a great opportunity to use USM - School of Chemical Sciences` corrosion labs.

Journal of Applied and Industrial Sciences, 2013, 1 (3): 78-88, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE)

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