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PHYTOCHEMICAL CONSTITUENTS OF THE LEAVES OF LEUCAENA LEUCOCEPHALA FROM MALAYSIA

Objective: This study was conducted to identify the phytochemical constituents of Leucaena leucocephala leaf extracts using gas chromatographymass spectrometry (GC-MS). Methods: Hexane, petroleum ether, chloroform, ethyl acetate and methanol leaves extract of L. leucocephala were analyzed using GC-MS, while the mass spectra of the compounds found in the extract were matched with the National Institute of Standards and Technology (NIST) library. Results: GC-MS analysis of L. leucocephala leaves revealed the presence of 30 compounds and the major chemical constituents were Squalene (41.02%), Phytol (33.80%), 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (30.86%) and 3,7,11-Tridecatrienenitrile, 4,8,12-trimethyl (25.64%). Some of these compounds have been reported to possess various biological activities such as antioxidant, antimicrobial, hepatoprotective, antiparasitic, insecticide, nematicide, pesticide, anti coronary, antiarthritic, antiandrogenic, hypocholesterolemic, cancer preventive, anti-cancer, analgesic, anesthetic, allergenic and etc. Conclusion: The findings of this study indicating that L. leucocephala leaves possess various potent bioactive compounds and is recommended as a plant of phytopharmaceutical importance.


INTRODUCTION
Leucaena leucocephala trees or Petai belalang as it is locally known in Malaysia [1] is a tropical multipurpose tree species. Central America and Southeast Asia's genus, Leucaena (English name), formerly contained approximately 50 species of both trees and shrubs that belonged to the Mimosoideae's subfamily of the Fabaceae family. L. leucocephala was known as miracle tree because of its worldwide success as a long-lived and highly nutritious forage tree, used as firewood, timber, human food, green manure, shade and erosion control and it is estimated to cover 2-5 million ha worldwide [2,3]. Almost every part of the L. leucocephala species is consumed as human food since the era of the Mayans [4]. In Indonesia, Thailand, and Central America, people eat the young leaves, flowers, and young pods in soups [5,6]. In addition, it is one of the medicinal plants used to control stomach ache, like contraception and abortifacient. Bioactivity studies on this plant revealed its anthelmintic, antibacterial, antiproliferative and antidiabetic activities [7].
To the best of our knowledge, since no information is available on the phytochemical screening of L. leucocephala leaves from Malaysia. Therefore, the present study was undertaken to investigate the extraction and phytochemical investigations of L. leucocephala leaves from Malaysia.

Plant material
L. leucocephala leaves were collected from Stutong located between 1° 31'8"N Latitude and 110° 22'41"E longitude in Kuching, Sarawak, Malaysia. The plant materials were taxonomically identified and confirmed by Dr. Mohamed Zayed, Forestry and Wood Technology Department, Faculty of Agriculture (EL-Shatby), Alexandria University, Alexandria, Egypt. Leaves were washed and dried at 30 °C in an oven until constant weights and ground to a powder with an electric blender.

Sample extraction
25 gm of the leaf powder of L. leucocephala for each solvent were weighted, transferred to a flask, treated with hexane, petroleum ether, chloroform, ethyl acetate and methanol until the powder was fully immersed and incubated overnight. The extracts were then filtered through Whatman filter paper No.1 along with 2 gm sodium sulfate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with sodium sulphate was wetted 95% ethanol. The filtrates were then air dried and subjected to gas chromatography-mass spectrometry analysis.

Gas chromatograph-mass spectroscopy (GC-MS)
GC-MS (Shimadzu QP 5000) was performed by using non-polar DB-5 cross-linked column (30 m long x 0.25 mm ID x 0.25 μm film thickness composed of 5% phenyl methyl polysiloxane). The initial temperature was programmed at 50 °C and held for two minutes, and then it was increased to 300 °C with the rate of 6.5 °C/min. The final temperature was held for ten minutes. The temperature of the injector and detector were set up to 280 °C and 300 °C, respectively. Helium gas was used as a carrier gas. 1 μl of the fractions was diluted in 100 μl hexane and then injected into the GC-MS [24][25]. Interpretation of mass-spectrum was conducted using the database of National Institute Standard and Technology (NIST). The spectrum of the unknown components was compared with the spectrum of known components stored in the NIST library. The name, molecular mass and structure of the components of the test materials were ascertained.
Phytol is one among the thirty compounds in L. leucocephala leaves which were also found to be effective at different stages of arthritis. It is used as preventive and therapeutic to against arthritis as well as it is a promising novel class of pharmaceuticals for the treatment of rheumatic arthritis and possibly other chronic inflammatory diseases [15,17,31]. In addition, it was observed to have antibacterial activities against Staphylococcus aureus by causing damage to cell membranes, as a result, there is a leakage of potassium ions from bacterial cells [14,32]. It is used along with simple sugar or corn syrup as a hardener in candies and it is a key acyclic diterpene alcohol that is a precursor for vitamins E and K [14].

CONCLUSION
The present phytochemical study of L. leucocephala leaf extracts was studied for the first time using five different solvent extracts with increasing polarities. In fact, this is the first available information about the phytoconstituents of L. leucocephala from Malaysia. L. leucocephala leaf extracts have revealed the presence of many secondary metabolites. The findings of this study confirmed that the L. leucocephala leaf extracts could be used as antioxidant, anticancer, cancer-preventive, diuretic, antiandrogenic, antimicrobial, antitumor, antiacne, antiarthritic, antieczemic, anti-inflammatory, hypocholesterolemic, insecticide, nematicide and pesticide. The present study, which reveals the presence of components in L. leucocephala leaf extracts suggest that the contribution of these compounds on the pharmacological activity should be evaluated.