LC-MS and GC-MS Profiling of Different Fractions of Ficus platyphylla Stem Bark Ethanolic Extract

The exploration of medicinal plants in traditional medicine for the treatment of diseases has been practiced for long, globally, because of its cultural acceptability, availability, and affordability. This study investigated the qualitative and quantitative estimation of phytochemicals present in Ficus platyphylla stem bark as well as determined the reducing power and antioxidant property of each fraction against DPPH and NO radicals. The study further elucidated the presence of possible compounds in different fractions (methanol, ethyl acetate, petroleum ether, and chloroform) of Ficus platyphylla stem bark (FPSB) extract using GC-MS, LC-MS, and FTIR techniques. Qualitative phytochemical analysis reveals the presence of phytochemicals: saponin, flavonoids, tannins, phenols, steroids, alkaloids, and glycoside in the ethanolic extract. The LC-MS study of methanol and ethyl acetate fractions reveals the presence of thirteen and three compounds, respectively. GC-MS analysis shows the presence of trans-13-octadecenoic acid as the main compound 38.07% and cis-vaccenic acid as the least compound (0.10%) in the petroleum ether fraction. The main compound in the chloroform fraction is 12-oleanen-3-yl acetate, (3. alpha.) with a peak area percentage of 49.25% and oleic acid been the least compound with 0.07% peak area. The FTIR analysis reveals that the fractions contain compounds with hydroxyl, aromatic, methyl, methylene, methyne, long aliphatic chain, ethers, ether-oxy, peroxides, etc. The analyzed fractions reveal compounds with potential pharmacological activity in the management of pathological conditions.


Introduction
Phytomedicine is a prominent form of traditional medicine, which has been in practice since time immemorial across the globe as a therapeutic approach to manage specifc pathological conditions [1]. Phytomedicine has been a culturally acceptable procedure to ameliorate many ailments because of its afordability and availability. Tis involves the use of herbal materials that may contain whole plant or its parts, which is known to enclose certain components that may afect the changes in the human body. Tus, various plants have been exploited for numerous purposes in the life of mankind and animals, explicitly, as food for nutritional benefts and medicines for the treatment of diseases [2]. Plants synthesize numerous chemical components called phytochemicals. Phytochemicals are non-nutritive bioactive components that have disease-preventive properties [3]. Tey are basically classifed into six major categories on the basis of their chemical structures and characteristics, namely carbohydrates, lipids, phenolics, terpenoids, alkaloids, and other nitrogen-containing compounds [4]. Tese categories are further divided via biogenesis to obtain alkaloids, saponins, glycosides, lignans, favonoids, tannins, triterpenes, coumarins, carotenoids, etc., which acts individually or synergistically to exhibit a useful or harmful efect to the body [4]. Tese phytochemicals are known to perform various functions and have been established to possess myriad of biological activities ranging from antibacterial, antifungal, antiviral, antioxidant, anticancer, hepatoprotective, antitumor benefts, and so much more. Modern drug research utilizes ethnobotany to search for pharmacologically active components (phytochemical) in plants, in order to alleviate serious side efects associated with synthetic drugs.
Ficus platyphylla is described as a deciduous plant belonging to the Moraceae family and usually grows in the tropical regions of world, basically in West Africa. In Nigeria, it is widely grown in the Northern part of the country. Te tree grows to a height of about 60 fts. In Hausa language, the tree is locally referred to as "Gamji" and belongs to the family of fg trees [5]. Traditionally, diferent parts of the plant such as the leaves, stem, bark, and root are believed by the Hausa communities in Northern Nigeria to be efcacious in the treatment of diferent ailments such as psychosis, infammation, epilepsy, and depression [5]. However, the stem bark of Ficus platyphylla has been predominantly used in Hausa traditional folk medicine to manage convulsive disorders. Phytochemical study from previous research has confrmed the presence of favonoids, tannins, and saponins in Ficus platyphylla stem bark [6]. Ficus platyphylla was reported to possess antioxidant, antiinfammatory, anti-insomnia, hepatoprotective, wound healing, and analgesic activities [7,8].
To ascertain the efcacy of a specifc medicinal plant to manage specifc ailments, one of the fundamental processes to undertake is extraction of phytochemicals contained in the plant. Plant extract usually contains several chemical constituents working synergistically to act as medicines (phytopharmaceuticals), toxins (phytotoxins), or both (phytopharmatoxins) [9]. Plant extracts can be obtained using selective solvent (polar, intermediate polar, and nonpolar) through various standard extraction procedures ranging from decoction, infusion, maceration, percolation, hot continuous extraction (Soxhlet), sonication, microwave-assisted extraction (MAE), supercritical fuid extraction, etc [10]. High content of polyphenolic components in medicinal plant has been related to their antioxidant efect that are vital in prevention of the development of age-related diseases, specifcally those associated with oxidative stress [7]. Te determination for a good procedure during extraction highly depends on the type of plant material, chemical properties of the solvent used, physical parameters of the experiment, and the intended use of fnal products [8]. Te investigation of the therapeutic efect of plants is vital for the exploration of medicinal plants; however, the determination of the essential active ingredients in a plant material can be overwhelming. Terefore, the study qualitatively and quantitatively assessed the presence of phytochemicals, determined the antioxidant property of each fraction against DPPH and NO radicals as well as reducing power activity, and identifed bioactive compounds present in four diferent fractions of Ficus platyphylla stem bark.

Preparation of Plant Material.
Te stem bark of the plant was dried under shade for fourteen days and pulverized using a pestle in a mortar to fne powder. Tis was subsequently stored in a labelled airtight container.

Preparations of Crude Extract of Ficus platyphylla Stem
Bark. Extraction was achieved using microwave-assisted extraction (MAE) as reported by Hossain et al. [11]. Briefy, 1000 g of pulverized Ficus platyphylla stem bark was dissolved in 3500 mL of 70% ethanol. Te solution was microwaved for two minutes at 80°C. After cooling, the extract obtained was fltered using a Whatman flter paper (No. 41). Te obtained fltrate was concentrated using a rotary evaporator.
2.6. Test for Alkaloids. Ethanolic extract of Ficus platyphylla stem bark (0.4 g) was stirred with 8 mL of 1% HCl, and the mixture was warmed and fltered. Te fltrate (2 mL) was treated separately with potassium bismuth (Dragendrof's reagent). Turbidity or precipitation with either of this reagent was taken as evidence for existence of alkaloids.

Test for Saponins.
Ethanolic extract of Ficus platyphylla stem bark (20 mg) was boiled in 20 ml of distilled water for 5 min and fltered. Te fltrate (10 mL) was mixed with 5 mL of distilled water. Te obtained mixture was vigorously shaken for froth formation. Tree drops of olive oil was mixed with froth, shaken vigorously, and observed for emulsion development.

Test for Terpenoids.
Presence of terpenoids was screened for using 5 mL (1 mg/mL) of ethanolic extract of Ficus platyphylla stem bark and mixed with 2 mL of chloroform, followed by 3 mL of concentrated H 2 SO 4 . A reddish-brown coloration of the interface confrmed the presence of terpenoids.
2.9. Test for Anthraquinones. Ethanolic extract of Ficus platyphylla stem bark (200 mg) was boiled with 6 mL of 1% HCl and fltered. Te fltrate was shaken with 5 mL of benzene, fltered, and 2 mL of 10% ammonia solution was added to the fltrate. Te mixture was shaken and the presence of a pink, violet, or red colour in the ammoniacal phase indicated the presence of free hydroxyl anthraquinones.

Cardiac
Glycosides. Presence of cardiac glycoside was screened for using 5 mL (10 mg/mL in methanol) of ethanolic extract of Ficus platyphylla stem bark mixed with 2 mL of glacial acetic acid (having a drop of FeCl 3 solution). To the mixture obtained, 1 mL of concentrated H 2 SO 4 was added to form a layer. Te presence of brown ring of the interface indicated deoxy sugar characteristic of cardiac glycosides.

Test for Flavonoids.
Ethanolic extract of Ficus platyphylla stem bark (50 mg) was suspended in 100 mL of distilled water to get the fltrate. A diluted ammonia solution (5 mL) was added to 10 ml of obtained fltrate, followed by few drops of concentrated H 2 SO 4 . Presence of favonoids was confrmed by yellow coloration.

Test for Tannins.
Ethanolic extract of Ficus platyphylla stem bark (50 mg) was boiled in 20 mL of distilled water and fltered. A few drops of 0.1% FeCl 3 was added to the fltrate and observed for colour change. A brownish green or a blue-black coloration was taken as evidence for the presence of tannins.

Test for Steroids.
Ethanolic extract of Ficus platyphylla stem bark (0.30 g) was reconstituted in 20 mL of ethanol, the mixture was extracted for 2 hours. 5 mL of the extract was added to 2 mL acetic anhydride followed with 2 mL of concentrated H 2 SO 4 . A violet to blue or green colour change in sample(s) indicates the presence of steroids.
2.14. Test for Phenols. Ethanolic extract of Ficus platyphylla stem bark was treated with 3-4 drops of ferric chloride solution. Formation of bluish-black colour indicated the presence of phenols.

Preparations of Fractions from Ethanolic Extract of Ficus platyphylla Stem
Bark. Te crude extract was partially purifed by sequential extraction using diferent solvents with diferent (increasing) polarity starting from petroleum ether, to chloroform, ethyl acetate, and fnally methanol. Te fractionation was done using Soxhlet apparatus (temperature � 40°C, time � 2-3 hours) as described by Hossain et al. [11]. Tis was done by tying the dried ethanolic extract with a muslin cloth and then inserted into the thimble of the Soxhlet apparatus. Te petroleum ether was poured in the distillation fask below and allowed to heat at 40°C. Te vapour resulting from the distillation fask condenses in the thimble-holder and dissolves the dried crude extract tied inside the thimble. As soon as the condensed petroleum ether in the extraction chamber reaches the overfow level, the solution in the thimbleholder was aspirated by a siphon and returns in the distillation fask. Tis was continuously done, until all compounds soluble in that solvent was extracted out. Te sample (dried crude extract) was then removed, untied, and dried under air. Te aforementioned procedure was done for chloroform, ethyl acetate, and methanol. Te obtained fractions of petroleum ether, chloroform, ethyl acetate, and methanol were concentrated via rotary evaporator, and the dried residues of each fraction were subjected to quantitative phytochemical screening, antioxidant assays, and phytochemical identifcation techniques using standard procedures. Te ethyl acetate and methanol fraction were subjected to LC-MS analysis while the petroleum ether and chloroform were subjected to GC-MS analysis on the basis of the polarity of solvent and target compounds.

Total Phenol.
Te total phenolic content of the obtained fractions was spectrometrically analyzed using the Folin− Ciocalteu method [14]. Briefy, 100 μL of each fraction dissolved in methanol (0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) and standard gallic acid was mixed with 2 mL of 2% (w/v) sodium carbonate solution. Te mixture was incubated for 5 min, and afterwards, 100 μL of Folin− Ciocalteu reagent was added. Te mixture was kept for 30 min at 25°C for colour development.
Absorbance was subsequently measured at 750 nm using a spectrophotometer. Results were expressed as mg/g of gallic acid equivalents (GAE) of dried Ficus platyphylla stem bark fraction.

Total Flavonoid.
Te total favonoid content of each fraction was determined using the aluminium chloride colorimetric assay with slight modifcations reported by Abdulqayoom et al. [15], and quercetin was used as a standard to construct the calibration curve. Quercetin (25 mg) was dissolved in 25 mL of aqueous ethanol (1 mg/mL stock solution) and then diluted to 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL with ethanol. About 20 μL each of the diferent fractions (0.1 g in 10 mL aqueous ethanol) and standard solution (0.2 to 1.0 mg/ mL) were mixed with 15 μL of sodium nitrite (0.5% NaNO 2 , w/v) solution separately in a 96 well plate and incubated for 6 min at room temperature (25°C). Tereafter, 15 μL of (1% AlCl 3 , w/v) solution was added to each reaction well and allowed to stand for further 6 min before the addition of 80 μL of sodium hydroxide (0.4% NaOH, w/v) to each well. Te mixtures were incubated for another 15 min at room temperature (25°C), and absorbance was taken at 510 nm. Te amount of favonoid was calculated from linear regression equation obtained from the quercetin standard calibration curve. Te favonoid content was calculated as mean ± SD (n � 3) and expressed as mg/g of quercetin equivalent of dried Ficus platyphylla stem bark fraction.

Antioxidant Assays of Petroleum Ether, Chloroform, Ethyl Acetate and Methanol Fraction of Ficus platyphylla Stem Bark
ing Activity Assay. Te DPPH radical scavenging assay was conducted in accordance to the method reported by Zhu et al. [16]. Briefy, 2 mL of DPPH solution (0.1 mM, in methanol) was mixed with 2 mL of the four diferent extracts at varying concentrations of 20, 40, 60, 80, 100, 120, and 140 μg/mL. Te reaction mixture was shaken and incubated in the dark at 25°C for 30 min. Te absorbance was read at 517 nm against the blank. Ascorbic acid was used as positive controls and prepared in a similar manner, as for the test samples. Te inhibition of the DPPH radical by the sample was calculated based on the following formula: Te half-maximal inhibitory concentration (IC 50 ) was measured to indicate the concentration required to inhibit DPPH radicals by half, and it was derived from the graph equation obtained from plot of respective concentration of standard ascorbic acid and each fraction (petroleum ether, chloroform, ethyl acetate, and methanol) against their obtained percentage inhibition.
Te IC 50 value is calculated from the graph equation

Nitric Oxide Inhibition Assay.
Te assay was conducted as reported by Fadzai et al. [17]. Te stock of each fraction and ascorbic acid was prepared (100 mg/mL) in methanol. Tese were then serially diluted to make concentrations of 20, 40, 60, 80, 100, 200, and 400 μg/mL. Griess reagent was prepared by mixing equal amounts of 2% sulphanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride immediately before use. A volume of 50 μL of 10 mM sodium nitroprusside (0.29 g/100 mL) in 0.1 M phosphate bufered saline was mixed with 50 μL of the diferent concentrations prepared in 96 well plate and then incubated at 25°C for 180 min. 100 μL of Griess reagent was added to the solution mentioned above. A control sample without the extracts but with an equal volume of methanol was prepared in a similar manner as was done for the test samples. Te absorbance was measured at 542 nm. Te percentage nitrite radical scavenging activity of the respective fractions and ascorbic acid was calculated using the following formula:

Reducing Power Assay.
According to the method reported by Nayan et al. [18], the aliquots of various concentrations of ascorbic acid (standard) and test samples (20 to 400 μg/mL) were dissolved in 1 mL of deionized water, which was then mixed with 2.5 mL of (pH 6.6) phosphate bufer and 2.5 mL of (1%) potassium ferricyanide. Te mixture was incubated at 50°C in water bath for 20 min after cooling. Aliquots of 2.5 mL of (10%) trichloroacetic acid were added to each of the mixture, which was then centrifuged at 3000 rpm for 10 min. Te upper layer of 2.5 mL of solution was mixed with 2.5 mL of distilled water and a freshly prepared 0.5 mL of (0.1%) ferric chloride solution. Te absorbance was measured at 700 nm. A blank was prepared without adding extract.

Identifcation of Ethyl Acetate and Methanol Fraction of FPSB Using Liquid Chromatography Mass Spectroscopy (LC-MS).
Te LC Waters e2695 separation module with W2998 PDA coupled to ACQ-QDA MS was used for this study. Te ethyl acetate and methanol fraction of FPSB was analyzed using an LC tandem MS as described by Piovesana et al. with slight modifcation [19]. Reconstitution of the fractions was done using methanol. Te fltration was carried out using polytetrafuoroethylene (PTFE) membrane flter (0.45 μm size). Ten microliters (10 μL) of the fltrate were introduced (injected) into the liquid chromatographical system. Tis was separated on Sunfre C 18

Statistical Analysis.
Analyses were carried out in triplicates using SPSS version 16 (IBM Inc. USA) and values were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to determine the level of signifcance at 95% confdence interval followed by Tukey's multiple comparison test. Te resolution quality of fgures [2][3][4][5] was harnessed using an Anguage digitizer to obtain the data points of both axis into an excel table and Ranalytic software was used in plotting the fgures.

Results
Te result in Table 1 (Figure 1). Methanol fraction also showed a strong scavenging activity against NO (92.42 ± 0.08) at 20 μg/mL compared to other fractions as shown in the table given below, although being lower than ascorbic acid (96.48 ± 0.05) at 20 μg/mL (Table 3). Te reducing power assay reveals methanol fraction activity to exhibit high reduction of Fe 3+ to Fe 2+ (1.33 ± 0.03) compared to other fractions as shown in Table 4.  Tables 5 and 6 show the identifed compounds in methanol and ethyl acetate fraction, respectively.

GC-MS Analysis of Petroleum Ether and Chloroform
Fraction. Te petroleum ether fraction reveals thirty-four compounds and eighteen compounds in the chloroform fraction as shown in Tables 7 and 8, respectively.  Figure 7 reveal that the methanol fraction contains a complex molecule. Te peak contains a single bond area (2500-4000 cm − 1 ), and at 3213 cm − 1 , it reveals a wider absorption band revealing a hydrogen bond in the   revealing an alcohol-hydroxy compound and aromatic nitro compound present, respectively. Same observation was made at 1520 cm − 1 . In the fngerprint region (400-1500 cm − 1 ), an aromatic compound was present at       Journal of Analytical Methods in Chemistry (400-1500 cm − 1 ), bands of 1454 cm -1, 1364 cm − 1 , and 1174 cm − 1 reveal saturated aliphatic (methylene), aliphatic nitro compounds, and aromatic rings. 1244 cm − 1 peak, 1095 cm − 1 peak, and 1025 cm − 1 peak reveal an aromatic ethers, cyclic ethers, and ether-oxy compounds while 879 cm − 1 peak, 808 cm − 1 peak, and 723 cm − 1 peak reveals a peroxides, aromatic ring, and phenyl compounds, respectively.

Discussion
Plants are made up of various chemical constituents which are reported to be biologically active and are responsible for exhibiting ranges of pharmacological activities. Much of these secondary metabolites present in plant are sources of natural antioxidants with reported safety level over synthetic ones [23]. Te radical scavenging activity of each fraction against DPPH and NO radicals was statistically signifcant at P < 0.05, with methanol fraction showing a strong antioxidant capacity compared to ethyl acetate, petroleum ether, and chloroform in respect to their IC 50 values. It was observed that the antioxidant capacity of each fraction occurs in a dose-dependent manner in proportion to increasing concentration. Te reducing power assay shows the activity of methanol fraction to reduce Fe 3+ to Fe 2+ as shown by increase in absorbance of the reaction mixture in proportion to increase in concentration. Te high total favonoid and total phenolic content of methanol fraction is evident that the fraction contained hydroxyl groups that confers sufcient antioxidant activity against DPPH and NO radicals which is in accordance to the report made by Hassan et al. [24]. Te study utilized the previous analytical techniques to identify diferent compounds present in the respective fractions on the basis of the nature of solvent as well as nature of target compound. Te LC-MS investigation of methanol fraction reveals the presence of thirteen compounds and three compounds in ethyl acetate fraction, respectively, as shown in Tables 5 and 6. However, a favonoid identifed as astilbin was present in the methanol fraction    and has been established to possess certain biological functions ranging from antioxidant, antifungal, anti-carcinogen, and anticonvulsant properties [25]. Te petroleum ether fraction analyzed via GC-MS identifed thirty-four compounds as listed in Table 7 with trans-13-octadecenoic acid being the main compound 38.07% and cis-vaccenic acid being the least compound 0.10%. One of the identifed phytochemicals, n-hexadecenoic acid has been reported in a previous study to possess an antioxidant, antibacterial, and antifungal property [26,27]. 9, 12-Octadecadienoic acid was reported to possess anti-infammatory and antibacterial properties and also used in beauty and skin care products [28]. Squalene has antioxidant, chemo-preventive activity against colon cancer, and anti-infammatory properties [26,27]. To elaborate further, hexadecenoic acid is found mostly in plants, animals, or micro-organism as a form of saturated fatty acid. It is used as release agents, soap production, and cosmetics. Methyl esters are found in pheromones and essential oils and are also used as fragrance [29]. Oleic acid and tripalmitin have anticholesterolemic, anti-infammatory, antifungal, antioxidative, and antibacterial properties [30]. Another compound of importance identifed is Cis-vaccenic acid. Tis is an omega 7 fatty acid reported to decrease LDLcholesterol and improve insulin sensitivity [31]. In addition, 9,12-octadecadienoic acid (Z,Z) methyl ester is a potent antioxidant that helps in prevention of prostate cancer disease, Alzheimer disease, and cardiovascular diseases [31].
Te chloroform fraction revealed eighteen compounds, as shown in Table 8 with 12-Oleanen-3-yl acetate (3.alpha.) being the main compound 49.25% and beta-sitosterol being the least compound 0.07%. Beta-sitosterol acts in declining the passage of cholesterol content in the blood vessels through the inhibition of cholesterol absorption at the digestive track. It is also very essential in other body processes due to its anti-infammatory properties and improves kidney functions. Lanosterol is utilized to alleviate lens opacity in age-related cortical cataract [32]. Te compounds identifed in Table 7 have common biological activity which include anti-infammatory, anti-bacterial, anti-fungal, antioxidant, anti-coronary, anti-acne, and anti-eczemic properties [33]. Te FTIR analysis of ethyl acetate fraction contains compound with a hydroxyl group, an aromatic ring, a long saturated aliphatic chain, nitro compounds, a double bond, absence of triple bond, an aldehyde, ethers, and peroxide components. Te methanol fraction contained a hydroxyl group, an aromatic ring, a long saturated aliphatic chain, absence of triple bond and ethers, and related components. Te petroleum ether fraction contained ether, aromatic ring, a long aliphatic chain (methyl and methylene), absence of triple bond, a phenyl, carbonyl, nitro, and peroxide components. Te chloroform fraction contained a long aliphatic chain (methyl and methylene), a carbonyl, phenyl, ether-oxy and an aromatic ring in the molecule. Te previous correlations were made in correspondence to the frequency range and functional group assignment reported by Nandiyanto et al. [34].

Conclusion
Te search for lead compounds from natural sources in the management of several pathological conditions is endless. Te outcome of this study reveals the antioxidant capacity of the respective fractions analyzed, as well as possible compounds present, whose bioactivity has been elucidated and reported and some to be scientifcally exploited in the future. It is established in this study that the methanol fraction contained poly-hydroxyl compounds compared to the other fractions analyzed, which must have contributed to its high antioxidant activity, thus making it a phytopharmacological agent to study other biological conditions such as aging, neurodegenerative disorders, cancer, and diabetes.

Data Availability
All data used are available in the manuscript.

Conflicts of Interest
Te authors declare that they have no conficts of interest.