Abstract: Background and Objectives: Phytocomponents from plant materials are largely responsible for their biologic activities. The phytocomponents from petroleum ether fraction of the leaf extract of P. guajava was identified, quantified and characterized. Materials and Methods: Chromatographic and spectrophotometric methods, namely, Gas chromatography-mass spectrometry (GC-MS) and Fourier transform-infrared spectrometry (FT-IR) were used for the identification, quantification and characterization of the phytocomponents. Results: The major phytocomponent from petroleum ether fraction of the leaf extract of P. guajava was bis (2-ethylhexyl) phthalate, whereas the minor phytocomponents were 2-pentadecanol and 14-pentadecenoic acid. Petroleum ether fraction of leaf extract of P. guajava gave a characteristic broad peak around 3328.5 cm–1, which was indicative of the presence of an alcohol functional group. Conclusion: GC-MS and FT-IR analyses of petroleum ether fraction of leaf extract of P. guajava identified five phytocomponents viz., 2-pentadecanol, carbonic acid, eicosyl vinyl ester, bis (2-ethylhexyl) phthalate, 14-pentadecenoic acid and 2-methyltetracosane.
INTRODUCTION
Guava (Psidium guajava L.), belongs to the family Myrtaceae, is a native of the American tropics from where it spread to the tropical and subtropical regions of the world. Guava tree bears fruits that are rich in ascorbic acid1. The plant is composed of medicinally relevant phytocomponents with variable pharmacological and clinical potentials2. According to Gutierrez et al.2 the biological activities of P. guajava leaf extract are consistent with the presence of notable phytocomponents such as the carotenoids, terpenoids, flavonoids, phenolics and triterpenes. Previous reports showed that aqueous extract of P. guajava was hepatoprotective3 and served as an expectorant4, whereas, the chloroform extract exhibited antimicrobial activities4.
Phytocomponents or bioactive principles are biomolecules that play major roles in therapeutic activities of herbs and are used in the pharmaceutical industries for the production of medicinal agents. Paradoxically, bioactive principles are also responsible for the toxic outcomes of some of these plants that are of concern and interest to the toxicologist5,6. Chromatographic and spectrophotometric protocols provide reproducible and dependable methods for the qualitative and quantitative evaluation as well as characterization of phytocomponents from medicinal plants7. Previous studies have reported the therapeutic potentials of several fractions of leaf extracts of P. guajava8 as well as toxic activities of petroleum ether fractions of plant extracts9. Because phytocomponents from plant materials are largely responsible for their biologic activities, the present investigation validates the identities, quantities and characteristics of phytocomponents from petroleum ether fraction of leaf extract of P. guajava. The phytocomponents from petroleum ether fraction of leaf extract of P. guajava were identified, quantified and characterized using chromatographic and spectrophotometric methods, namely, Gas chromatography-mass spectrometry (GC-MS) and Fourier transform-infrared spectrometry (FT-IR).
MATERIALS AND METHODS
Study area: The study was carried out at the Medical Biochemistry Laboratory, Department of Biochemistry, Imo State University, Owerri, Nigeria from April to September, 2019.
Collection of leaf samples: Healthy and matured leaves of P. guajava found growing within the location (Latitude 5°30.2237'N, Longitude 7°2.6277’E) were harvested during the wet season of 2nd and 7th April, 2019. The leaves were authenticated by a taxonomist. The voucher number of the leaves was IMSUH 010. For future reference purposes, a specimen of the leaves was deposited in the herbarium.
Extraction and preparation of fractionated of leaf extract: The extraction protocol, using the soxhlet extractor, was carried out within 24 h of collection of the leaf samples of P. guajava according to the methods previously described by Chikezie et al.10. Fractionation of the leaf extract was according to the methods of Okoye et al.11 whereby the crude hydro-ethanolic leaf extract was partitioned with an equal volume of petroleum ether. The petroleum ether fraction of the leaf extract was finally concentrated under reduced pressure for 24 h at 50°C in a rotary evaporator (BüchRotavapor R-200). The petroleum ether fraction was subjected to GC-MS and FT-IR analyses.
GC-MS and FT-IR analyses: Chromatographic-spectrophotometric protocols were carried out using GC-MS systems {Agilent 7890A GC system set up with 5975C VL MSD, Agilent Technologies, Inc., Santa Clara, CA, USA; The MS system was accomplished in electron ionization (EI) mode with Selected Ion Monitoring (SIM)}12. FT-IR instruments (PerkinElmer Spectrophotometer, USA) protocols were according to the methods previously described by Ighodaro et al.13.
RESULTS
Phytocomponent composition of P. guajava extract by GC-MS chromatogram: Phytocomponents from petroleum ether fraction of leaf extract of P. guajava are presented in Table 1. The major phytocomponent from petroleum ether fraction of the leaf extract was bis (2-ethylhexyl) phthalate, which constituted 71.49% in relative abundance compared to other phytocomponents (Table 1). Additionally, the minor phytocomponents, in terms of their relative abundance, were 2-pentadecanol (2.52%) and 14-pentadecenoic acid (2.35%) (Table 1).
Peak values of FT-IR spectra of P. guajava extract: The characteristic peak values of FT-IR spectra of petroleum ether fraction of leaf extract of P. guajava are summarized in Table 2. Petroleum ether fraction of leaf extract of P. guajava gave a characteristic broad peak around 3328.5 cm–1, which was indicative of the presence of an alcohol functional group. The weak band within the region of 2855.1-2922.2 cm–1 and 1315.8-1453.7 cm–1 were characteristic of alkane sp3 C-H bend.
| Table 1: | Phytocomponent composition of petroleum ether fraction of leaf extract of P. guajava by GC-MS chromatogram |
| RT: Retention time, MF: Molecular formula, MW: Molecular weight, PA: Peak area | |
| Table 2: | Peak values of FT-IR spectra of petroleum ether fraction of leaf extract of P. guajava |
| T: Transmittance | |
The presence of nitrogen-containing functional groups, namely, the cyanates (C≡N) and isothiocyanates (-NCS) were characterized by weak bands around the regions of 2117.1-2292.3 cm–1.
The presence of carbonyl and anhydride functional groups were typified by weak bands around the regions of 1718.3-1871.1 cm–1. The peak at 1613.9 cm–1 was indicative of the presence of aromatic compounds in petroleum ether fraction of P. guajava. Finally, other notable phytocomponents from petroleum ether fraction of leaf extract of P. guajava were acyl and phenol (1233.7 cm–1), secondary amines and cyanates (1162.9 cm–1) and alkoxyl (1028.7 cm–1) functional group-containing compounds.
DISCUSSION
Phytocomponents exhibit medicinal and toxic activities that are consistent with their sources, molecular and chemical peculiarities5. The phytocomponents from petroleum ether fraction of leaf extract of P. guajava appeared to justify its biological and medicinal activities reported elsewhere2,14-18. Empirical investigations had revealed a connection between the length of the carbon chain of aliphatic alcohols and their anti-bacterial activities19,20. FT-IR analysis of petroleum ether fraction of leaf extract of P. guajava confirmed the presence of aliphatic alcohols such as 2-pentadecanol. Previous reports precisely showed that n-pentadecanol, an analog of 2-pentadecanol, was the phytocomponent from methanolic flower extract of Saussurea obvallata that exhibited anti-oxidant and anti-microbial properties in vitro21.
Likewise, bis (2-ethylhexyl) phthalate from seed extract of Buchholzia Coriacea Engler (Capparaceae)22, flower extract of Calotropis gigantea (Linn)23 and Penicillium janthinellum24 62 exhibited antioxidant, antitumor, antiviral, anti-fungal and anti-bacterial properties. The present study revealed that bis (2-ethylhexyl) phthalate was a major phytocomponent from petroleum ether fraction of leaf extract of P. guajava, which validates its antioxidant, anti-fungal and anti-bacterial properties when juxtaposed with previous research findings14,15,17,18,23,25. In addition, reports showed that bis (2-ethylhexyl) phthalate (di-2-ethylhexyl phthalate) including structurally similar molecular analogs such as the dicarboxylic acids viz. 1, 2-cyclohexanedicarboxylic acid and phthalic acid analogs stabilized erythrocyte membrane against osmotic stress in vitro26,27.
The aforementioned beneficial properties notwithstanding, a wide range of toxic outcomes of bis (2-ethylhexyl) phthalate following continuous exposure of human population to environmental products and contaminants has been extensively reviewed28. From a generalized viewpoint, the issues of beneficial and toxic properties as well as the nature and origin of bis (2-ethylhexyl) phthalate in biologic systems are still controversially discussed23,28,29.
The presence of 14-pentadecenoic acid analogs, pentadecenoic acid and its various isomers, in folklore medicinal plant (Exacum lawii) has been reported by Sharma and Hemalatha30. Carbonic acid, eicosyl vinyl ester, by virtue of its antioxidant activity, was among the phytocomponents from Cakile maritima Scop extracts that inhibited the growth of some bacteria, which triggered autoimmune inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis and multiple sclerosis31. The presence of phenols, mono- and di-carboxylic acids and their ester derivatives, including nitrogen-containing functional group compounds, were expressly confirmed by FT-IR spectra patterns. Accordingly, the presence of carbonic acid, eicosyl vinyl ester in P. guajava validates its use in the treatment of bacterial infections as previously reported by Sanda et al.15, Rahman et al.32, Morais-Braga et al.33 and Diaz-de-Cerio et al.34.
Studies have revealed that 2-methyltetracosane is a free radical scavenging phytocomponent from whole plant methanol extract of Cenchrus ciliaris35. Fittingly, in concord with previous reports by Sanda et al.15, Diaz-de-Cerio et al.34 and Joseph and Priya36, the application of P. guajava decoction by traditional practitioners in ameliorating pathologic states, induced by oxidative stress, appeared to be connected with the 2-methyltetracosane content of petroleum ether fraction of leaf extract of P. guajava.
Further studies aimed at isolating the phytocomponents from petroleum ether fraction of leaf extract of P. guajava as well as ascertaining their capacities to ameliorate pathologic conditions associated with oxidative stress are recommended.
CONCLUSION
GC-MS and FT-IR analyses of petroleum ether fraction of leaf extract of P. guajava identified five phytocomponents viz., 2-pentadecanol, carbonic acid, eicosyl vinyl ester, bis (2-ethylhexyl) phthalate, 14-pentadecenoic acid and 2-methyltetracosane. The major phytocomponent from petroleum ether fraction of the leaf extract was bis (2-ethylhexyl) phthalate, whereas, the minor phytocomponents include 2-pentadecanol and 14-pentadecenoic acid. Some of these phytocomponents have been reported to exhibit biologic and therapeutic activities.
SIGNIFICANCE STATEMENT
This study discovered that notable phytocomponents from petroleum ether fraction of leaf extract of P. guajava, namely, bis (2-ethylhexyl) phthalate, 2-pentadecanol and 14-pentadecenoic acid can be of potential benefit for the treatment of viral, fungal and bacterial infections as well as amelioration of pathologic conditions linked with oxidative stress. This study will help the researcher to uncover the critical area of the use of the identified phytocomponents from petroleum ether fraction of leaf extract of P. guajava for the treatment of pathologic conditions that many researchers were not able to explore.
ACKNOWLEDGMENTS
The authors are grateful for the technical assistance offered by Mr. F.C. Emengaha, Chief Academic Technologist, Department of Medical Biochemistry, College of Medicine and Mr. C.O. Kabiri, Senior Laboratory Technologist, Department of Biochemistry, Faculty of Science, Imo State University, Owerri. The efforts of Mr. Franklyn O. Ohiagu are highly appreciated. This work was supported by Imo State University, Owerri and research grant offered by the Tertiary Education Trust Fund (TETFund) Research Based Interventions of Nigerian Universities. Imo State University, Owerri, provided the laboratory space and infrastructures. TETFund provided the financial resources for purchase of laboratory chemicals/reagents and instruments as well as expenses pertaining to transportation and travels. Grant Number: TETFUND/DRSS/UNIV/OWERRI/2015/5RP VOL 1 (7).