Antioxidant and Antimicrobial Activities of β-Caryophyllene dominated Leaf Essential oil of Trichilia monadelpha (Thonn.)

Trichilia monadelpha (Thonn) JJ De Wilde (Meliaceae) is an essential plant of diverse medicinal uses in folk medicine. It is deployed in the treatment of ulcers/epidermal infections, epilepsy, gonorrhea and inflammatory disorders such as arthritis etc. This study investigated the leaf essential oil constituents of T. monadelpha along with the corresponding antioxidant and antimicrobial activities. The essential oil of fresh leaves of T. monadelpha was isolated by hydrodistillation using all glass Clevenger distillation apparatus while the identification and characterization was done by Gas Chromatography-Mass Spectrometry (GC-MS). Antioxidant activity was carried out by scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) at 517nm in a UV-Visible spectrophotometric assay with Ascorbic acid, Butylated hydroxylanisole (BHA) and α-Tocopherol as reference standards. Antimicrobial assay was examined by Agar diffusion method. A total of fifteen compounds were identified in the essential oil with β-Caryophyllene (35.41%) as the major constituent. The oil demonstrated moderate inhibition against seven of the ten microorganisms used for the assay, namely: Staphylococcus aureus, Esherichia coli, Bacillus subtilis, Pseudomonas aerugunosa, Candida albicans, Aspergillus niger, Rhizopus stolonifer. The antioxidant activity of the essential oil (IC50 of 3.72 mg/ml) though lower than that of ascorbic acid (IC50 of 1.47 mg/ml) and BHA (IC50 of 1.88 mg/ml) was however better than that of α-Tocopherol (IC50 of 4.83 mg/ml).The results obtained corroborated the uses of Trichilia monadelpha in folk medicine.


Introduction
Plants have been the major source in the treatment of various diseases, especially in developing countries (Benoit-Vical, 2006). Some of these plant extracts and phytochemicals are known to have antimicrobial properties and are of great significance in therapeutic treatments. A number of studies have been conducted in different countries to demonstrate such efficacy (Senatore et al, 2007;Langseth et al, 1996). Medicinal plant constituents have a long history of use in old and modern medicines. They are the source of important medications such as vincristine, codeine, quinine, morphine, digoxin, atropine and others, some of which are still currently in use (Raskin et al, 2002).
Essential oils from aromatic plants and spices have a long history of use as natural microbial agents in folk medicine. They also have wide applications in food, cosmetics and pharmaceutical industries as these oils are known to possess antimicrobial, antiviral and antioxidant properties. Essential oils are employed as antioxidants due to their small molecular size and their ability to easily penetrate the skin tissue. They are lipid soluble and are capable of penetrating the membranes easily even in conditions where oxygen deficiency leads to hardening of membranes. Studies reveal that essential oils serve as powerful antioxidants that produce adverse environment for damaging free radicals, thus preventing mutations and oxidants in cells. They therefore function as scavengers for free radicals. Essential oils are extracted from different plant parts: fruits, leaves, flowers, barks, roots and seeds with each part possessing unique characteristics (Bako et al, 2005;Bray, 1999;Potterat, 1997).
Oxidative damage in human cells or tissues is mainly due to the uncontrolled production of oxygen from free radicals. These are harmful substances produced in the body during the normal metabolic processes. Bacterial, fungal and viral infections have also been reported to produce reactive oxygen species. Environmental chemicals such as pesticides and other chemicals like alcohol, cigarette smoking, burning of organic matter, UV radiations and automobile pollution also contribute to free radical generation (Sato et al, 1995). Medicinal plants with antibacterial and antioxidant potentials are thus used as alternative therapy for bacterial infections and many kind of degenerative diseases in folk medicine (Cai, 1996).
However, the need for new antioxidant and antimicrobial agents has increased remarkably over the years due to multiple drug resistance and fewer side effects of drugs from natural sources as compared to synthetic drugs. T. monadelpha (Thonn) JJ De Wilde (Meliaceae) is a medium-sized tree that grows 12-20 m high, distributed in deciduous and semi-deciduous secondary forests, often in wet places in West Africa. T. monadelpha has diverse application in folklore in the treatment of ailments namely: Yaws, Ulcers, abortion, arthritis, cough, gonorrhea, syphilis, chancres and syphilistic sores, wounds/cuts/epidermal infections, rickets, sterility, amenorrhea, stomach pain, gastrointestinal pain and disorders, dysentery and fever (Burkill, 1997;Abbiw, 1990).
In a continuing search for more potent natural products with fewer side effects Oloyede et al., 2020;Onocha et al., 2016), we present the result of the investigation of the chemical constituent, antioxidant and antimicrobial activities of the leaf essential oil of T. monadelpha which is being reported for the first time.

Plant material
Fresh leaves of T. monadelpha were collected from botanical garden of University of Ibadan, Ibadan. Specimens were identified and authenticated at Forest Research Institute of Nigeria (FRIN), Oyo State, Nigeria where a voucher specimen with herbarium number FHI 112655 was deposited in the FRIN herbarium. The leaves were chopped and hydro-distilled immediately.

Isolation of essential oil
The oil was obtained by hydro-distillation on all glass Clevenger distillation apparatus for 4hrs in accordance with the British pharmacopoeia specifications (1980). The essential oil (TML) was collected and stored at 4℃ prior to analysis.

Gas Chromatography-Mass Spectrometry
GC-MS analyses of the essential oil was analyzed on an Aligent Technologies 7890A GC system coupled to a 5975c VLMSD mass spectrometer with an injector 7683b series device. An Agilent (9091)-413:325℃ HP-5 column (30 m ӿ 320 µm ӿ 0.25 µm) was used with helium as carrier gas at flow rate of 3.3245 mL min -1 . The GC oven temperature was initially programmed at 50℃ (hold for 1 min) and finally at 300℃ (hold for 5mins) at a rate of 80℃ min -1 while the trial temperature was 37.25℃. The column heater was set at 250℃ and was a split less mode while the pressure was 10.153 psi with an average velocity of 66.45 cm sec -1 and a hold-up time of 0.75245 min was recorded. Mass spectrometry was run in the Electron Impact mode (EI) at 70 eV. The percentage compositions were obtained from electronic integration measurements using Flame Ionization Detector (FID) set at 250℃. The peak numbers and relative percentages of the characterized components are given in Table 1.
Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.11, No.16, 2020 37 2.6 Antioxidant Activity A 0.5 ml of the radical source of 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) solution in methanol was prepared and 3 ml of this solution was mixed with 1 ml of the oil sample in methanol Onocha et al., 2011). The decrease in absorption (A) at 517 nm of DPPH was measured after 10 mins of incubation. The actual decrease in absorption was measured against that of control and the percentage inhibition (% I) was also calculated. The same experiment was carried out on ascorbic acid, α-Tocopherol and butylated hydroxylanisole (BHA) which are known antioxidants. All test and analysis were run in triplicates and the result obtained were averaged. The activities were determined as a function of their % inhibition which was calculated using the formula: % = × 100 2.7 Antimicrobial Activity All microorganisms were clinical isolates obtained from the Department of Pharmaceutical Microbiology, University of Ibadan, Nigeria. The leaf essential oil of T. monadelpha was tested against 10 strains of microorganisms consisting of 7 bacteria: 5 Gram negative (Escherichia coli and Klebsiella pneumonia, Salmonella typhi, Klebsiellae pneumonia, Pseudomonas aeruguinosa), 2 Gram-positive: (Staphylococcus aureus and Bacillus subtilis) and 3 fungi (Candida albicans, Penicillum notatum and Aspergillus niger). Agar diffusion method (Pour broth dilutions and surface plate for antibacterial and antifungal activities, respectively) was employed (Kalemba & Kunicka, 2003;Okpala et al., 2019). Oil sample was prepared such that 1 ml of the oil was regarded as 100 % concentration. 0.5 ml into 0.5 ml of Hexane gave 50 % concentration and 25 % concentration was obtained using serial dilution. Hexane was used as a negative control, Gentamicin (10 µg/mL) as a positive control for bacteria and Tioconazole (0.7 mg/mL) as positive control for Fungi. All tests were carried out in triplicates. Observed zones of inhibition of growth were measured and recorded in millimetre (mm).
Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.11, No.16, 2020  3.2 Antioxidant Activity Antioxidant activity (scavenging assay) of essential oil of T. monadelpha leaves was determined with DPPH method. The scavenging assay was carried out to determine the ability of the essential oil in inhibiting free radicals.
The leaf essential oil of T. monadelpha displayed a moderate free radical scavenging activity at all the concentrations varying from 60.5 % to 53.6% as shown in Table 2. The antioxidant activity of the essential oil though lower than that of ascorbic acid and BHA was however better than that of α-Tocopherol. Furthermore, the scavenging activity effectiveness was measured through IC50 (the concentration of the samples required to scavenge 50 % of the hydroxyl radicals. The lower the IC50, the greater the overall effectiveness of the suspected antioxidant potency. From the results obtained, the antioxidant effectiveness is as follows: ascorbic acid (1.47 mg/ml) > butylated hydroxylanisole (1.88 mg/ml) > TML (3.72 mg/ml) > α-Tocopherol (4.83 mg/ml). The percentage inhibition is presented in Table 2 and Fig. 1.  Vol.11, No.16, 2020 Fig 1: % Inhibition of leaf essential oil (TML) and standards

Conclusion
Fifteen compounds were identified in the essential oil from the leaves of T. monadelpha wherein β-Caryophyllene was the most abundant compound. The predominance of the bioactive β-Caryophyllene which is known for its  Vol.11, No.16, 2020 diverse pharmacological properties: antioxidant, wound healing, analgesic, anti-inflammatory, anti-anaphylactic, antidepressant and antimicrobial, in the leaf essential oil could be responsible for the diverse medicinal application of the plant in folk medicine. The results obtained justifies the uses of Trichilia monadelpha in folk medicine.