Determination of bioactive constituents of Spondias mombin leaves by GC-MS analysis

The therapeutic capabilities of abundant herbal products have been known. However, further researches are required to properly screen, isolate and probably characterize their various active components. In this study, an attempt was made to identify the compounds present in the ethanol and n-hexane extracts of S. mombin leaves using gas chromatography and mass spectrometry (GC-MS) technique. Spondias mombin leaves were collected, air dried and pulverized into powdery form and extracted with ethanol and n-hexane at room temperature. The ethanol and n-hexane extracts of S. mombin leaves were analyzed by GC-MS. The GC-MS analysis of the ethanol extract of S. mombin leaves showed twenty-five (25) compounds, while twenty-nine (29) compounds were identified in n-hexane extract of S. mombin leaves. However, both extracts had ten compounds in common: n-Hexadecanoic acids, Phytol, Phenol 3pentadecyl-, Phenol 3-methyl-, Phenol 2-methyl-, 2H-1-Benzopyran-6-ol, Gamma-Tocopherol, Vitamin E, Lup-20(29)en-3-one and Stigmast-4-en-3-one. The percentage proportion of these compounds differed in each extract. In view of this, the possible biological properties of these identified compounds suggested that S. mombin might possibly be potential therapeutic agents.


Introduction
Traditional medicine has remained a great alternative basis of medicine worldwide, particularly in low income countries. It has been stated that 80 % of the world populace (about 4 billion people) are dependent mainly on herbal therapies and traditional therapeutic practice for their well-being care (Ekor, 2014). The increasing occurrence of resistance (particularly to antibiotics), detrimental side effects, cost of accessible therapies and the well understanding of illness conditions has led to a renewed curiosity in the development of newer molecules from natural bases (Cowan, 1999). Therefore, aggressive screening of flora used in traditional medicine as well as an attempt to recognize and undertake chemical clarification of the compounds accountable for their stated activities are been undertaken by researchers (Elufioye and Berida, 2018).
Gas Chromatography Mass Spectrometry (GC-MS) is one of the compatible techniques advantageous in recognizing the bioactive components of long chain, alcohols, branched chain hydrocarbons, esters, acids, etc present in crude plant materials (Sathiyabalan et al., 2014). Gas chromatography (GC) is broadly used for compound analysis, drug screening and assessment resulting from environmental pollution. Mass spectrometry (MS) measures the mass-to-charge ratio of ions of analysts and is a plot of strength as a function of mass-to-charge ratio (Kim et al., 2005;Shah et al., 2019). Relying on the database of National Institute of Standards and Technology Chemistry Web Book (2019) for diverse compounds, researchers can thus recognize and quantify these potential compounds and components.
Amongst numerous known therapeutic plants is Spondias mombin. Spondias mombin L. (Family-Anacardiaceae) also recognized as Hog plum, is a plant that grows in nearly every part of the world. It is a deciduous tree that produces many fruits which is about 20 m tall and it grows in the rain forest and the coastal part of Africa. It is recognized locally as "iyawe" and "iyeye" by the Hausa and Yoruba people of Nigeria respectively (Dharmananda, 2003). Tradomedicine physicians across Africa use all parts of the plant for therapeutic purposes. The fruits' decoction is drunk as a febrifuge and diuretic, while the decoction of the leaves and stem-bark are used as an emetic, dysentery and anti-diarrhea (Osuntokun et al., 2018). Spondias mombin is a good recipe for the treatment of gonorrhea and leucorrhea as well as for haemorrhoids (Li et al., 2003). A tea of the leaves and flowers of S. mombin is taken to relieve stomach ache, biliousness, cystitis, urethritis, throat and eye inflammations. Herbalists in Southwest Nigeria use the plant in the treatment of diabetics, psychiatric disorders, typhoid, nervous disorders and tuberculosis (Newman et al., 2008). The extract of the powder of the dried leaves and fresh crushed leaves are used for healing inflammation, wounds, varicose ulcers, burn and frost-bite in herbal medicine (Shaarawy et al., 2009). The bark, fruits and leaves of S. mombin are said to be rich in antioxidants and other valuable phytochemicals (Igwe et al., 2011;Omoregie and Oikeh, 2015). However, much work has not been done on the identification of the compounds accountable for these stated activities. Using GC-MS, we aimed to identify the compounds present in the ethanol and n-hexane extracts of S. mombin leaves.

Collection of Plant Sample
Spondias mombin leaves were obtained from the Faculty of Pharmacy's premises, University of Benin, Benin City, Edo State, Nigeria and authenticated at the Department of Plant Biology and Biotechnology with voucher number UBH-S345. The plant was air dried under room temperature and pulverized into powdery form which was kept in an air-tight container ready for the analysis.

Plant extraction
Cold extraction method was used in this study, by dissolving the pulverized plant material in ethanol and n-hexane as solvents. The filtrate for each extraction was combined, and evaporated to dryness at room temperature.

Gas Chromatography -Mass Spectrometry (GC-MS)
GC-MS analysis of the ethanol and n-hexane extracts of S. mombin leaves were performed using Hewlett-Packard (HP) 6890 series (Agilent) Gas Chromatography System, interfaced to HP 5973 series (Agilent) mass spectrometer, equipped with an autosampler and a single capillary injector. TR-FAME (Thermo 260 M142P) (70 % cyanopropylpolysilphenylenesiloxane) capillary GC column (30 m x 0.25 mm, i.d., x 0.25 μm film thickness) was also used. This was done by dissolving the sample in the organic solvent till it dissolved completely. GC condition was maintained at 100 -280 °C as 5 °C/min. 2 μl of sample was injected into the column. The helium gas was allowed to move at 1 ml/min through the column and the compound split in the ratio of 1:10. After the program, mass spectrometer scanned the compounds separated and each peak area was measured to find the compounds present at the area. The results were represented as chromatogram graph in the GC-MS.

Results
The GC analysis and mass spectral data of the ethanol and n-hexane extracts of S. mombin leaves showing compound name, retention time, peak area (%), molecular weight (g/mol), structural formula, compound nature and structure of compound is presented in Tables 1 and 2. Twenty five (25) compounds were identified in the ethanol extract (Table 1), while twenty-nine (29) compounds were detected in the n-hexane extract of the S. mombin leaves ( Table 2). Figures 1 and 2 depict the chromatograms of ethanol and n-hexane extracts of S. mombin leaves respectively with their peak areas. In ethanol extract, phenol, 2-methyl-gave the highest peak at 21.918 retention time ( Figure 1) while phenol, 3-methylgave the highest peak at 21.941 retention time in n-hexane extract ( Figure 2).

Discussion
The determination of phytochemicals in useful and regularly consumed plant products is significant. Thus, using gas chromatography and mass spectrometry (GC-MS) method, this study attempted to identify and quantify compounds present in the ethanol and n-hexane extracts of S. mombin leaves. Twenty five (25)  It has been reported that squalene acts as a drug transporter by chemically linking with medicines to advance certain physicochemical properties. For instance, administration of squalene-doxorubicin nanohybrids caused higher decrease of pancreatic cancers when related with free doxorubicin (Wicki et al., 2015). Consequently, squalene based nanoparticles have been deliberated to be likely candidates for anticancer medications (Kotelevets et al., 2017;Saha et al., 2015). In the nutritious context, virgin olive oil is a vital basis of squalene (Beltrán et al., 2016). Intake of olive oil has been interrelated with lower risk of tumor growth in several cancer kinds (Newmark, 1999;Lozano-Grande et al., 2018). Kalvodona (2010) stated that squalene is not very vulnerable to peroxidation and seems to function in the skin as a quencher of daylight, guarding human skin surface from fat peroxidation owing to contact to UV and other sources of ionizing emission.
Also, 9,12 Octadecadienoic acids identified in n-hexane extract of S. mombin leaves in this study had been determined in methanol extract of Nymphaea lotus and S. mombin to have antimicrobial activity (Odumosu et  Remarkably, the key isolated compound from the petroleum ether extract of Abutilon indicum was recognized as beta-sitosterol, which introduces this natural compound as a unique mosquito larvicidal sterol.

Conclusion
In conclusion, the study had been able to determine the possible chemical constituents of the ethanol and n-hexane extracts of S. mombin leaves using GC-MS technique. A number of compounds identified were found in abundance in the extracts. However, the percentage proportion of these compounds differed in each extract. In view of this, the possible biological properties of these identified compounds suggested that S. mombin might possibly be potential therapeutic agents.

Acknowledgments
The author recognized the technical skills of Mr. Isaac at the Central Laboratory of Federal University of Technology, Akure (FUTA).

Disclosure of conflict of interest
The author declared no conflict of interest.