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Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 6349332, 14 pages https://doi.org/10.1155/2022/6349332 Research Article LC-MS and GC-MS Profiling of Different Fractions of Ficus platyphylla Stem Bark Ethanolic Extract 1 2 3 4 Madinat Hassan , Sunday Zeal Bala , Musa Bashir, Peter Maitalata Waziri, 5 2 6 Ramlatu Musa Adam, Muhammad Abdullahi Umar, and Priscilla Kini Biology Department, Faculty of Science, Airforce Institute of Technology, Kaduna State, Nigeria Biochemistry Department, Faculty of Basic Medical Science, Bayero University Kano, Kano State, Nigeria Centre for Dryland Agriculture, Faculty of Agricultural Science, Bayero University Kano, Kano State, Nigeria Biochemistry Department, Faculty of Science, Kaduna State University, Kaduna State, Nigeria Biochemistry Department, Faculty of Science, Gombe State University, Gombe State, Nigeria Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Correspondence should be addressed to Priscilla Kini; email@example.com Received 14 September 2021; Revised 29 July 2022; Accepted 17 August 2022; Published 14 December 2022 Academic Editor: Radosław Kowalski Copyright © 2022 Madinat Hassan et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te 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 afordability. Tis study investigated the qualitative and quantitative es- timation 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. Te study further elucidated the presence of possible compounds in diferent 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, favonoids, tannins, phenols, steroids, alkaloids, and glycoside in the ethanolic extract. Te 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. Te 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. Te FTIR analysis reveals that the fractions contain compounds with hydroxyl, aromatic, methyl, methylene, methyne, long aliphatic chain, ethers, ether-oxy, peroxides, etc. Te analyzed fractions reveal compounds with potential pharmacological activity in the management of pathological conditions. have been exploited for numerous purposes in the life of 1. Introduction mankind and animals, explicitly, as food for nutritional Phytomedicine is a prominent form of traditional medicine, benefts and medicines for the treatment of diseases . which has been in practice since time immemorial across the Plants synthesize numerous chemical components called globe as a therapeutic approach to manage specifc patho- phytochemicals. Phytochemicals are non-nutritive bioactive logical conditions . Phytomedicine has been a culturally components that have disease-preventive properties . acceptable procedure to ameliorate many ailments because Tey are basically classifed into six major categories on the of its afordability and availability. Tis involves the use of basis of their chemical structures and characteristics, namely herbal materials that may contain whole plant or its parts, carbohydrates, lipids, phenolics, terpenoids, alkaloids, and which is known to enclose certain components that may other nitrogen-containing compounds . Tese categories afect the changes in the human body. Tus, various plants are further divided via biogenesis to obtain alkaloids, 2 Journal of Analytical Methods in Chemistry saponins, glycosides, lignans, favonoids, tannins, tri- 2. Materials and Methods terpenes, coumarins, carotenoids, etc., which acts individ- 2.1.CollectionofPlantMaterial. Ficusplatyphylla (Moracea) ually or synergistically to exhibit a useful or harmful efect to stem bark was obtained from a village called Karau-Karau, the body . Tese phytochemicals are known to perform located in Zaria, Kaduna state, Nigeria with GPS co-ordi- various functions and have been established to possess nates; 11.061516, 7.716590, on 24th November, 2019. myriad of biological activities ranging from antibacterial, antifungal, antiviral, antioxidant, anticancer, hep- atoprotective, antitumor benefts, and so much more. 2.2. Identifcation of Plant Material. Identifcation and au- Modern drug research utilizes ethnobotany to search for thentication of the plant material was carried out at the pharmacologically active components (phytochemical) in Biology Department, Ahmadu Bello University, Zaria, plants, in order to alleviate serious side efects associated Kaduna State by the botanist Mr. U.S. Gallah. A sample with with synthetic drugs. voucher number 7719 was deposited in the Herbarium. Ficus platyphylla is described as a deciduous plant be- longing to the Moraceae family and usually grows in the 2.3.PreparationofPlantMaterial. Te stem bark of the plant tropical regions of world, basically in West Africa. In was dried under shade for fourteen days and pulverized Nigeria, it is widely grown in the Northern part of the using a pestle in a mortar to fne powder. Tis was subse- country. Te tree grows to a height of about 60 fts. In Hausa quently stored in a labelled airtight container. language, the tree is locally referred to as “Gamji” and be- longs to the family of fg trees . Traditionally, diferent parts of the plant such as the leaves, stem, bark, and root are 2.4. Preparations of Crude Extract of Ficus platyphylla Stem believed by the Hausa communities in Northern Nigeria to Bark. Extraction was achieved using microwave-assisted be efcacious in the treatment of diferent ailments such as extraction (MAE) as reported by Hossain et al. . Briefy, psychosis, infammation, epilepsy, and depression . 1000 g of pulverized Ficus platyphylla stem bark was dis- However, the stem bark of Ficus platyphylla has been pre- solved in 3500 mL of 70% ethanol. Te solution was dominantly used in Hausa traditional folk medicine to ° microwaved for two minutes at 80 C. After cooling, the manage convulsive disorders. Phytochemical study from extract obtained was fltered using a Whatman flter paper previous research has confrmed the presence of favonoids, (No. 41). Te obtained fltrate was concentrated using a tannins, and saponins in Ficus platyphylla stem bark . rotary evaporator. Ficus platyphylla was reported to possess antioxidant, anti- infammatory, anti-insomnia, hepatoprotective, wound 2.5. Qualitative Phytochemical Screening. Te phytochemi- healing, and analgesic activities [7, 8]. cal constituents of Ficus platyphylla stem bark (FPSB) was To ascertain the efcacy of a specifc medicinal plant to screened qualitatively for presence or absence of favonoids manage specifc ailments, one of the fundamental processes to (lead acetate test), phenols (ferric chloride test), saponin undertake is extraction of phytochemicals contained in the (foam test), tannin (gelatin test), alkaloids (Dragendorf’s plant. Plant extract usually contains several chemical constit- reagent), cardiac glycosides, anthraquinones, and terpenoids uents working synergistically to act as medicines (phyto- using methods described by Saeed et al. . Steroid was pharmaceuticals), toxins (phytotoxins), or both screened for using the method described by Ezeonu and (phytopharmatoxins) . Plant extracts can be obtained using Ejikeme . selective solvent (polar, intermediate polar, and nonpolar) through various standard extraction procedures ranging from decoction, infusion, maceration, percolation, hot continuous 2.6.TestforAlkaloids. Ethanolic extract of Ficus platyphylla extraction (Soxhlet), sonication, microwave-assisted extraction stem bark (0.4 g) was stirred with 8 mL of 1% HCl, and the (MAE), supercritical fuid extraction, etc . High content of mixture was warmed and fltered. Te fltrate (2 mL) was polyphenolic components in medicinal plant has been related treated separately with potassium bismuth (Dragendrof’s to their antioxidant efect that are vital in prevention of the reagent). Turbidity or precipitation with either of this re- development of age-related diseases, specifcally those associ- agent was taken as evidence for existence of alkaloids. ated with oxidative stress . Te determination for a good procedure during extraction highly depends on the type of 2.7. Test for Saponins. Ethanolic extract of Ficus platyphylla plant material, chemical properties of the solvent used, physical stem bark (20 mg) was boiled in 20 ml of distilled water for parameters of the experiment, and the intended use of fnal 5 min and fltered. Te fltrate (10 mL) was mixed with 5 mL products . Te investigation of the therapeutic efect of of distilled water. Te obtained mixture was vigorously plants is vital for the exploration of medicinal plants; however, shaken for froth formation. Tree drops of olive oil was the determination of the essential active ingredients in a plant mixed with froth, shaken vigorously, and observed for material can be overwhelming. Terefore, the study qualita- emulsion development. tively and quantitatively assessed the presence of phyto- chemicals, determined the antioxidant property of each fraction against DPPH and NO radicals as well as reducing 2.8.TestforTerpenoids. Presence of terpenoids was screened power activity, and identifed bioactive compounds present in for using 5 mL (1 mg/mL) of ethanolic extract of Ficus four diferent fractions of Ficus platyphylla stem bark. platyphylla stem bark and mixed with 2 mL of chloroform, Journal of Analytical Methods in Chemistry 3 followed by 3 mL of concentrated H SO . A reddish-brown 2.14. Test for Phenols. Ethanolic extract of Ficus platyphylla 2 4 coloration of the interface confrmed the presence of stem bark was treated with 3-4 drops of ferric chloride terpenoids. solution. Formation of bluish-black colour indicated the presence of phenols. 2.9. Test for Anthraquinones. Ethanolic extract of Ficus platyphylla stem bark (200 mg) was boiled with 6 mL of 1% 2.15.PreparationsofFractionsfromEthanolicExtractofFicus HCl and fltered. Te fltrate was shaken with 5 mL of platyphylla Stem Bark. Te crude extract was partially benzene, fltered, and 2 mL of 10% ammonia solution was purifed by sequential extraction using diferent solvents added to the fltrate. Te mixture was shaken and the with diferent (increasing) polarity starting from petroleum presence of a pink, violet, or red colour in the ammoniacal ether, to chloroform, ethyl acetate, and fnally methanol. phase indicated the presence of free hydroxyl Te fractionation was done using Soxhlet apparatus anthraquinones. (temperature � 40 C, time � 2-3 hours) as described by Hossain et al. . Tis was done by tying the dried ethanolic extract with a muslin cloth and then inserted into 2.10. Cardiac Glycosides. Presence of cardiac glycoside was the thimble of the Soxhlet apparatus. Te petroleum ether screened for using 5 mL (10 mg/mL in methanol) of etha- was poured in the distillation fask below and allowed to nolic extract of Ficus platyphylla stem bark mixed with 2 mL heat at 40 C. Te vapour resulting from the distillation fask of glacial acetic acid (having a drop of FeCl solution). To the condenses in the thimble-holder and dissolves the dried mixture obtained, 1 mL of concentrated H SO was added to 2 4 crude extract tied inside the thimble. As soon as the form a layer. Te presence of brown ring of the interface condensed petroleum ether in the extraction chamber indicated deoxy sugar characteristic of cardiac glycosides. reaches the overfow level, the solution in the thimble- holder was aspirated by a siphon and returns in the dis- 2.11. Test for Flavonoids. Ethanolic extract of Ficus platy- tillation fask. Tis was continuously done, until all com- phylla stem bark (50 mg) was suspended in 100 mL of dis- pounds soluble in that solvent was extracted out. Te tilled water to get the fltrate. A diluted ammonia solution sample (dried crude extract) was then removed, untied, and (5 mL) was added to 10 ml of obtained fltrate, followed by dried under air. Te aforementioned procedure was done few drops of concentrated H SO . Presence of favonoids 2 4 for chloroform, ethyl acetate, and methanol. Te obtained was confrmed by yellow coloration. fractions of petroleum ether, chloroform, ethyl acetate, and methanol were concentrated via rotary evaporator, and the dried residues of each fraction were subjected to quanti- 2.12. Test for Tannins. Ethanolic extract of Ficus platyphylla tative phytochemical screening, antioxidant assays, and stem bark (50 mg) was boiled in 20 mL of distilled water and phytochemical identifcation techniques using standard fltered. A few drops of 0.1% FeCl was added to the fltrate and procedures. Te ethyl acetate and methanol fraction were observed for colour change. A brownish green or a blue-black subjected to LC-MS analysis while the petroleum ether and coloration was taken as evidence for the presence of tannins. chloroform were subjected to GC-MS analysis on the basis of the polarity of solvent and target compounds. 2.13. 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 2.16. Percentage Yield of Extract. Te percentage yield of added to 2 mL acetic anhydride followed with 2 mL of ethanolic extract and other fractions of Ficus platyphylla concentrated H SO . A violet to blue or green colour change stem bark was obtained after drying in the water bath for 2 4 in sample(s) indicates the presence of steroids. 48 h at 40 C and was calculated as (Weight of extract) Yield of extract(%) � × 100. (1) (Weight of dry plant before extraction) standard gallic acid was mixed with 2 mL of 2% (w/v) sodium 3. Quantitative Phytochemical Screening of carbonate solution. Te mixture was incubated for 5 min, and Petroleum Ether, Chloroform, Ethyl Acetate afterwards, 100μL of Folin− Ciocalteu reagent was added. Te and Methanol Fraction of Ficus platyphylla mixture was kept for 30 min at 25 C for colour development. Stem Bark Absorbance was subsequently measured at 750 nm using a spectrophotometer. Results were expressed as mg/g of gallic 3.1. Total Phenol. Te total phenolic content of the obtained acid equivalents (GAE) of dried Ficus platyphylla stem bark fractions was spectrometrically analyzed using the Folin− Ciocalteu method . Briefy, 100 μL of each fraction fraction. dissolved in methanol (0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) and 4 Journal of Analytical Methods in Chemistry 3.2. Total Flavonoid. Te total favonoid content of each (n � 3) and expressed as mg/g of quercetin equivalent of dried fraction was determined using the aluminium chloride col- Ficus platyphylla stem bark fraction. orimetric assay with slight modifcations reported by Abdulqayoom et al. , and quercetin was used as a standard 4. Antioxidant Assays of Petroleum Ether, to construct the calibration curve. Quercetin (25 mg) was Chloroform, Ethyl Acetate and Methanol dissolved in 25 mL of aqueous ethanol (1 mg/mL stock so- Fraction of Ficus platyphylla Stem Bark lution) and then diluted to 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL with ethanol. About 20μL each of the diferent fractions (0.1 g in 4.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scaveng- 10 mL aqueous ethanol) and standard solution (0.2 to 1.0 mg/ ing Activity Assay. Te DPPH radical scavenging assay was mL) were mixed with 15μL of sodium nitrite (0.5% NaNO , conducted in accordance to the method reported by Zhu w/v) solution separately in a 96 well plate and incubated for et al. . Briefy, 2 mL of DPPH solution (0.1 mM, in 6 min at room temperature (25 C). Tereafter, 15 μL of (1% methanol) was mixed with 2 mL of the four diferent extracts AlCl , w/v) solution was added to each reaction well and at varying concentrations of 20, 40, 60, 80, 100, 120, and allowed to stand for further 6 min before the addition of 80μL 140μg/mL. Te reaction mixture was shaken and incubated of sodium hydroxide (0.4% NaOH, w/v) to each well. Te in the dark at 25 C for 30 min. Te absorbance was read at mixtures were incubated for another 15 min at room tem- 517 nm against the blank. Ascorbic acid was used as positive perature (25 C), and absorbance was taken at 510 nm. Te controls and prepared in a similar manner, as for the test amount of favonoid was calculated from linear regression samples. Te inhibition of the DPPH radical by the sample equation obtained from the quercetin standard calibration was calculated based on the following formula: curve. Te favonoid content was calculated as mean ± SD (Absorbance of control) − (Absorbance of sample) (2) % Inhibition � × 100. (Absorbance of control) Te half-maximal inhibitory concentration (IC ) was 20, 40, 60, 80, 100, 200, and 400μg/mL. Griess reagent was measured to indicate the concentration required to inhibit prepared by mixing equal amounts of 2% sulphanilamide in DPPH radicals by half, and it was derived from the graph 5% phosphoric acid and 0.1% naphthyl ethylenediamine equation obtained from plot of respective concentration of dihydrochloride immediately before use. A volume of 50μL standard ascorbic acid and each fraction (petroleum ether, of 10 mM sodium nitroprusside (0.29 g/100 mL) in 0.1 M chloroform, ethyl acetate, and methanol) against their ob- phosphate bufered saline was mixed with 50 μL of the tained percentage inhibition. diferent concentrations prepared in 96 well plate and then Te IC value is calculated from the graph equation incubated at 25 C for 180 min. 100μL of Griess reagent was Y �MX − C, Where Y � 50, X � IC value, M � coefcient of added to the solution mentioned above. A control sample X and C � constant. 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 4.2. Nitric Oxide InhibitionAssay. Te assay was conducted percentage nitrite radical scavenging activity of the re- as reported by Fadzai et al. . Te stock of each fraction spective fractions and ascorbic acid was calculated using the and ascorbic acid was prepared (100 mg/mL) in methanol. following formula: Tese were then serially diluted to make concentrations of Absorbance of Control − Absorbance of Sample (3) % Inhibition � × 100. Absorbance of Control 4.3. Reducing Power Assay. According to the method re- cooling. Aliquots of 2.5 mL of (10%) trichloroacetic acid ported by Nayan et al. , the aliquots of various con- were added to each of the mixture, which was then centrations of ascorbic acid (standard) and test samples (20 centrifuged at 3000 rpm for 10 min. Te upper layer of to 400μg/mL) were dissolved in 1 mL of deionized water, 2.5 mL of solution was mixed with 2.5 mL of distilled water which was then mixed with 2.5 mL of (pH 6.6) phosphate and a freshly prepared 0.5 mL of (0.1%) ferric chloride so- bufer and 2.5 mL of (1%) potassium ferricyanide. Te lution. Te absorbance was measured at 700 nm. A blank was mixture was incubated at 50 C in water bath for 20 min after prepared without adding extract. Journal of Analytical Methods in Chemistry 5 4.4. Identifcation of Ethyl Acetate and Methanol Fraction of carried out to prepare the translucent sample discs. Te FPSB Using Liquid Chromatography Mass Spectroscopy (LC- samples (400 to 4000 scan range) were loaded into the FTIR − 1 MS). Te LC Waters e2695 separation module with W2998 spectroscope, with 4 cm resolution for each fraction. PDA coupled to ACQ-QDA MS was used for this study. Te ethyl acetate and methanol fraction of FPSB was analyzed using 4.7. Statistical Analysis. Analyses were carried out in trip- an LC tandem MS as described by Piovesana et al. with slight licates using SPSS version 16 (IBM Inc. USA) and values modifcation . Reconstitution of the fractions was done using were expressed as mean± standard deviation. One-way methanol. Te fltration was carried out using polytetra- analysis of variance (ANOVA) was used to determine the fuoroethylene (PTFE) membrane flter (0.45 μm size). Ten level of signifcance at 95% confdence interval followed by microliters (10μL) of the fltrate were introduced (injected) into Tukey’s multiple comparison test. Te resolution quality of the liquid chromatographical system. Tis was separated on fgures [2–5] was harnessed using an Anguage digitizer to Sunfre C 5.0μm 4.6 mm × 150 mm column. Te run was obtain the data points of both axis into an excel table and R- performed at a fow rate of 1.0 mL/min. Te temperature of the analytic software was used in plotting the fgures. column was fxed at 25 C with a 0.1% formic acid mixed in water serving as solvent A in the mobile phase. Also, formic acid (0.1%) 5. Results was mixed with acetonitrile serving as solvent B within the gradient. A ration of 95 : 5 (A : B) was maintained for another Te result in Table 1 reveals the phytochemical constituent 1 minute. 5 : 95(A : B) for 13–15 minutes, 95 : 5 (A : B) to 17, 19 of Ficus platyphylla stem bark ethanolic extract (70% aq.). and 20 minutes. Setting the PDA detector at 210–400 nm, 1.2 nm resolution and10 points/sec sampling rate. Scan range fromm/z 5.1.TotalPhenolandTotalFlavonoid. Te methanol fraction 100–1250 was used to acquire mass spectra maintaining the of Ficus platyphylla stem bark contained the highest fa- following settings: ESI source in both positive and negative ion vonoid content at concentration of 527.38 mg compared to modes; 600 C probe temp, 10 mL/min fow rate, and 45 psi ethyl acetate (512.74 mg), chloroform (274.89 mg), and nebulizer gas. Te MS was set with a fragmentation voltage of petroleum ether fractions (126.49 mg). Te highest total 125 V in an automatic mode. Processing of data generated was phenol content was observed in methanol fraction with a done using Empower 3. Te following information was used in concentration of 92.46 mg compared to ethyl acetate the identifcation of the various compounds present in the plant (86.63 mg), chloroform (59.31 mg), and petroleum ether material, retention time (tR), elution order, fragmentation (7.02 mg) fractions, respectively. Te abovementioned de- pattern, and Base m/z. tails can be found in Table 2. 4.5. Gas Chromatography-Mass Spectroscopy Analysis of Pe- 5.2. Antioxidant Activity. Methanol fraction reveals the troleumEtherandChloroformFractions. Te petroleum ether highest scavenging activity against DPPH radicals (84.90± 0.05) and chloroform fractions were analyzed using GS-MS as de- at concentration of 140μg/mL with IC of 58.15μg/mL, al- scribed by Konappa et al. . Te GC system (PerkinElmer though less than the standard-ascorbic acid (93.85± 0.05) at Clarus 600) was close-ftting a Rtx-5MS capillary column. A concentration of 140μg/mL with IC of 16.93μg/mL (Figure 1). constant fow rate of 1.0 mL/min was maintained with helium Methanol fraction also showed a strong scavenging activity (99.99%) as the carrier gas. Te method used for the detection of against NO (92.42± 0.08) at 20μg/mL compared to other the GC-MS spectral lines was the ionization energy method, fractions as shown in the table given below, although being lower with ionization energy of 70 eV (electron volt) and a 0.2 seconds than ascorbic acid (96.48± 0.05) at 20μg/mL (Table 3). Te scan time with a ranging fragment from 40 to 600 m/z, with one reducing power assay reveals methanol fraction activity to ex- 3+ 2+ microliter (1μL) injection quantity (split ratio 10 :1) and a hibit high reduction of Fe to Fe (1.33± 0.03) compared to temperature maintained at 250 C. However, the column oven other fractions as shown in Table 4. ° ° temperature was at 50 C running for 3 minutes and 10 C temperature increase per minutes up to 280 C, with a fnal 5.3.LCMSAnalysisofMethanolandEthylAcetateFractionsof temperature of about 300 C for 10 minutes. Retention time per Ficus platyphylla Stem Bark (FPSB). LC-MS analysis was minute, peak area, peak height, and spectral lines patterns were conducted for methanol and ethyl acetate fraction of FPSB on used to identify the components present in the sample plant the basis of polarity of the solvent used. Te LC-MS analysis materials when compared with spectral lines from the database reveals the retention time of the possible compounds as shown of authenticated compounds stored in the National Institute of in Figures 2 and 3 and the fragmentation pattern reveal the Standards and Technology (NIST) library . relative abundance of the identifed compounds as shown in Figures 4 and 5. Tables 5 and 6 show the identifed compounds in methanol and ethyl acetate fraction, respectively. 4.6. Fourier-Transform Infrared Spectroscopy (FT-IR Agilent Carry 630). Protocol for FTIR spectroscopy was done as reported by Satapute et al.  which involves the encap- 5.4. GC-MS Analysis of Petroleum Ether and Chloroform sulation of 10 mg of the dried petroleum ether, chloroform, Fraction. Te petroleum ether fraction reveals thirty-four ethyl acetate, and methanol fractions in hundred milligrams compounds and eighteen compounds in the chloroform (100 mg) of potassium bromide (KBr) pellet. Tis was fraction as shown in Tables 7 and 8, respectively. 6 Journal of Analytical Methods in Chemistry Table 1: Phytochemical compounds of the aqueous ethanolic extract of FPSB. Constituent Qualitative analysis Saponins + Tannins + Alkaloids + Flavonoids + Cardiac glycosides + Phenols + Steroids + Anthraquinones − Terpenoids − Presence (+), Absence (− ). Table 2: Percentage yield (%w/w), total phenolic, and favonoid content of the diferent fractions obtained from ethanolic extract of Ficus platyphylla stem bark. Initial weight of dried Ficus platyphylla stem bark � 1000 g Plant extract Weight of recovered Percentage yield Total favonoid content (mg of QE/g Total phenol content (mg of GAE/g extract (g) (% w/w) dry weight of FPSB fraction) dry weight of FPSB fraction) FPSB crude 116.38 11.63 — — extract Petroleum 3.43 2.94 126.49± 0.004 7.02± 0.0145 ether Chloroform 2.68 2,30 274.89± 0.0213 59.31± 0.0004 Ethyl acetate 4.59 3.94 512.74± 0.0120 86.63± 0.0001 Methanol 81.04 69.63 527.38± 0.0040 92.46± 0.0003 Values are expressed as mean± SD of triplicate determination. Values are not statistically signifcant at P> 0.05. GAE- gallic acid equivalent, QE- quercetin equivalent, FPSB- Ficus platyphylla stem bark. − 1 The Percentage Inhibition of DPPH free radical scavenging 2855 cm revealing the presence of an aliphatic compound. Two peaks were observed at triple bond region − 1 − 1 (2000–2500 cm ), at frequency range of 2158 cm and − 1 2031 cm implying the presence of C�C bond in the form of nitrogen double bond and cumulated double bond com- pound and aromatic ring molecule, respectively. Regarding *87.13 − 1 the double bond region (1500–2000 cm ), narrow peak at *71.65 − 1 55 *16.93 *69.49 about 1733 cm attests the presence of a carbonyl com- pound, which could be aldehyde. A sharp bend was observed 45 *58.15 − 1 at 1606 cm revealing an aromatic ring stretch and aromatic nitro compounds present, respectively. Same observation − 1 was noticed at 1520 cm . In the fngerprint region Key: * IC − 1 (400–1500 cm ), an aromatic compound was present at − 1 − 1 20 40 60 80 100 120 140 strong visible bands of 767 cm for ortho and 816 cm for Concentration (mg/mL) para position on aromatic ring structure. A strong visible − 1 band was observed at 1144, 1095, and 1036 cm revealing Ethyle acetate Chloroform the presence of secondary amine, cyclic ethers, and ether- Methanol Ascorbic − 1 − 1 oxy compounds, respectively. Peaks at 1438 cm 1364 cm Petroluem Ether − 1 1244 cm reveal saturated aliphatic (methyl), aliphatic ni- Figure 1: Te percentage DPPH-free radical scavenging activity tro, and aromatic ether compounds, respectively. Te bands obtained from diferent fractions of ethanolic extract of Ficus − 1 − 1 at 875 cm and 723 cm reveal presence of peroxides and platyphylla stem bark. phenyl compounds as shown on the spectra. 5.5. FTIR Analysis of Ethyl Acetate Fraction of Ethanolic ExtractofFicusplatyphyllaStemBark. Te numerous peaks 5.6.FTIRAnalysisofMethanolFractionofEthanolicExtractof shown in Figure 6 reveal that the ethyl acetate fraction FicusplatyphyllaStemBark. Te numerous peaks shown in contained a complex molecule. Te peak contains a single Figure 7 reveal that the methanol fraction contains a − 1 bond area (2500–4000 cm ) with a wider absorption band complex molecule. Te peak contains a single bond area − 1 − 1 − 1 at 3216 cm . Tis indicates the presence of a hydrogen bond (2500–4000 cm ), and at 3213 cm , it reveals a wider in the molecule. Tere is a sharp band at around 2922 and absorption band revealing a hydrogen bond in the Percentage Inhibition (%) Journal of Analytical Methods in Chemistry 7 Table 3: Te percentage nitric oxide radical scavenging activity obtained from diferent fractions of ethanolic extract of Ficus platyphylla stem bark. % NO inhibition activity of diferent fractions of Ficus platyphylla stem bark Concentration of fractions of Ficus platyphylla stem bark Petroleum (μg/mL) Ethyl acetate Methanol Chloroform Ascorbic acid ether a a a a a 20 80.14± 0.04 92.42± 0.08 81.64 ± 0.02 77.60± 0.04 94.32± 0.03 b b a a a 40 79.46± 0.08 87.24± 0.07 80.06± 0.07 75.63± 0.04 90.22± 0.04 a ab a a c 60 72.84± 0.07 84.63± 0.05 75.95± 0.05 71.02± 0.04 84.39± 0.07 c c a b ab 80 71.228± 0.0 81.26 ± 0.09 71.46 ± 0.02 70.26± 0.06 82.84± 0.10 bc c a ab b 100 68.64± 0.05 77.88± 0.03 68.52± 0.03 69.62± 0.06 80.32± 0.04 c bc a c c 200 63.99± 0.04 73.01± 0.09 61.38 ± 0.07 67.24± 0.09 78.46± 0.07 d bcd b c d 400 61.34± 0.03 68.46± 0.04 57.90 ± 0.03 61.92± 0.04 72.78± 0.05 Values are expressed as mean± SD of triplicate determination. Values bearing diferent superscript are statistically signifcant at P< 0.05 (Tukey’s multiple comparison test). Table 4: Te reducing power activity obtained from diferent fractions of ethanolic extract of Ficus platyphylla stem bark. Reducing power (RP) activity of diferent fractions of Ficus platyphylla stem bark compared with ascorbic acid standard. Concentration of fractions of Ficus platyphylla stem bark (μg/mL) Petroleum Ethyl acetate Methanol Chloroform Ascorbic acid ether 20 0.32± 0.03 0.33± 0.01 0.28± 0.02 0.21± 0.01 0.34± 0.02 40 0.36± 0.02 0.38± 0.02 0.31± 0.01 0.23± 0.01 0.39± 0.03 60 0.44± 0.04 0.42± 0.04 0.33± 0.04 0.27± 0.01 0.45± 0.01 80 0.49± 0.01 0.46± 0.01 0.38± 0.02 0.28± 0.01 0.46± 0.03 100 0.50± 0.02 0.54± 0.02 0.42± 0.01 0.31± 0.01 0.72± 0.02 200 0.72± 0.03 0.99± 0.1 0.72± 0.01 0.58± 0.01 0.84± 0.01 400 0.90± 0.06 1.33± 0.03 0.88± 0.03 0.77± 0.02 1.24± 0.04 Values are expressed as mean± SD of triplicate determination. LCMS− Methanol Fraction Chromatogram 2.5 2.0 1.5 AU 1.0 0.5 0.0 2 4 6 8 101214 Retention time (min) Figure 2: LC-MS chromatogram of methanol fraction of FPSB ethanolic extract. − 1 molecule. No triple bond region (2000–2500 cm ) was revealing an alcohol-hydroxy compound and aromatic detected, implying the absence of C ≡ C bond in the nitro compound present, respectively. Same observation − 1 molecule. Regarding the double bond region was made at 1520 cm . In the fngerprint region − 1 − 1 − 1 (1500–2000 cm ), a sharp bend was observed at 1606 cm (400–1500 cm ), an aromatic compound was present at 2.440 5.026 5.752 6.110 7.014 9.571 9.831 10.679 11.329 12.754 14.615 14.947 15.038 8 Journal of Analytical Methods in Chemistry LCMS− Ethyl Acetate Fraction Chromatogram 0.4 0.3 AU 0.2 0.1 0.0 5 10 15 Retention time (min) Figure 3: LC-MS chromatogram of ethyl acetate fraction of FPSB ethanolic extract. Mass Spectra - Methanol Fraction Mass Spectra - Ethyl Acetate Fraction 461.54 599.09 199.51 500.01 599.79 196.05 274.51 418.39 219.46 601.34 214.47 367.22 475.6 289.12 390.38 0 100 200 300 400 500 600 0 100 200 300 400 500 600 m (z) m (z) Figure 4: Mass spectra of methanol fraction of FPSB ethanolic Figure 5: Mass spectra of ethyl acetate fraction of FPSB ethanolic extract. extract. − 1 − 1 contains a complex molecule. Te peak contains a single strong visible bands of 767 cm for phenyl and 857 cm − 1 bond area (2500–4000 cm ) and reveals the presence of an for para position on aromatic ring structure. A strong − 1 ether and oxy compound in the molecule. Tere is a sharp visible band was observed at 1438, 1341, and 1244 cm − 1 − 1 − 1 − 1 band at around 2922 and 2855 cm revealing the presence 1200 cm 1095 cm 1036 cm . Tis revealed saturated of an aliphatic compound (methyl and methylene). No triple aliphatic group (methyl and methyne), aromatic ethers, − 1 bond region (2000–2500 cm ) was detected, implying the aromatic ring, cyclic ether, and cyclo-hexane ring struc- absence of C ≡ C bond in the molecule, although transition tures, respectively. − 1 metal carbonyl was observed at band of 2094 cm . Re- − 1 garding the double bond region (1500–2000 cm ), a narrow − 1 peak at about 1733 cm , reveals a carbonyl compound, 5.7. FTIR Analysis of Petroleum Ether Fraction of Ethanolic − 1 which could be an aldehyde and at 1938 cm , informs ExtractofFicusplatyphyllaStemBark. Te numerous peaks presence of aromatic ring. In the fngerprint region shown in Figure 8 reveal that the petroleum ether fraction Intensity 1.7207 2.4134 5.743 6.2123 7.419 8.8268 9.8324 9.9665 Intensity 11.352 12.3128 12.7374 12.8268 13.8101 13.9665 14.4581 14.9721 15.0615 15.5307 16.6034 17.3184 18.0335 Journal of Analytical Methods in Chemistry 9 Table 5: LC-MS profle of compounds identifed from methanol fraction obtained from ethanolic extract of Ficus platyphylla stem bark. Retention time Max. intensity % Base peak Chemical Compound (min) (intensity) area (m/z) composition 1.750 1835.64 0.35 196.05 C H NO 2,4-Dimethoxyamphetamine 11 17 2 1.932 2647.32 0.22 214.47 C H NO 4-Phenoxyphenethylamine 14 15 2.622 1328.79 0.20 219.46 C H O 2-Octylphenylketone 15 22 4.986 2032.79 0.71 274.51 C H NO Ethanol, 2,2-(dodecylamino) bis- 16 35 2 5.752 1407126.77 1.80 289.12 C H O Triphenylacetic acid 20 16 2 9.571 7224.28 0.65 367.22 C H FO Methylmalonic acid, 2-fuorophenyloctylester 21 31 4 Isonipecotic acid, N-(4-fuoro-2- 9.831 158583.73 0.65 390.38 C H F NO 19 23 4 3 trifuoromethylbenzoyl)-, pentyl ester 11.329 214827.62 94.78 418.39 C H NO Sarcosine, N-(4-ethylbenzoyl)-dodecylester 24 39 3 12.755 3232.84 0.65 451.00 C H O Astilbin 21 22 11 14.948 3361.58 0.47 461.54 C H O Succinic acid, 3-ethylphenyl heptadecyl ester 29 48 4 15.045 5352.51 0.08 500.01 C H Triacontylbenzene 36 66 15.926 3892.86 1.96 599.79 C H O Si Octaethylene glycol, 2TBDMS derivative 28 62 9 2 17.364 6738.94 0.64 601.34 C H N O Nicofuranose 30 24 4 10 NIST standard reference database number 69. Table 6: LC-MS profle of compounds identifed from ethyl acetate fraction obtained from ethanolic extract of Ficus platyphylla stem bark NIST standard reference database number 69. Base peak Chemical Retention time (min) Max. intensity (intensity) % area Compound (m/z) composition 1.640 1465.86 0.23 199.51 C H ClO Butanoic acid, 4-chlorophenyl ester 10 11 2 1.824 2937.48 0.34 475.60 C H O Succinic acid, 3-ethylphenyl octadecyl ester 30 50 4 2.426 1316.62 0.42 599.09 C H O Si 1,2-Triacontanediol, di-TMS 36 78 2 2 − 1 - − 1 (400–1500 cm ), bands of 1454 cm 1, 1364 cm , and 6. Discussion − 1 1174 cm reveal saturated aliphatic (methylene), aliphatic − 1 Plants are made up of various chemical constituents which nitro compounds, and aromatic rings. 1244 cm peak, − 1 − 1 are reported to be biologically active and are responsible for 1095 cm peak, and 1025 cm peak reveal an aromatic exhibiting ranges of pharmacological activities. Much of ethers, cyclic ethers, and ether-oxy compounds while − 1 − 1 − 1 these secondary metabolites present in plant are sources of 879 cm peak, 808 cm peak, and 723 cm peak reveals a natural antioxidants with reported safety level over synthetic peroxides, aromatic ring, and phenyl compounds, ones . Te radical scavenging activity of each fraction respectively. against DPPH and NO radicals was statistically signifcant at P < 0.05, with methanol fraction showing a strong antioxi- dant capacity compared to ethyl acetate, petroleum ether, 5.8.FTIRAnalysisofChloroformFractionofEthanolicExtract and chloroform in respect to their IC values. It was ob- of Ficus platyphylla Stem Bark. Te chloroform fraction 50 served that the antioxidant capacity of each fraction occurs contains complex molecule as shown by the numerous in a dose-dependent manner in proportion to increasing peaks. Te peaks shown in Figure 9 contain a single bond − 1 concentration. Te reducing power assay shows the activity area (2500–4000 cm ) and reveal the presence of aliphatic 3+ 2+ − 1 of methanol fraction to reduce Fe to Fe as shown by chains (methyl and methylene) at 2922 cm and − 1 increase in absorbance of the reaction mixture in proportion 2855 cm , respectively. No triple bond region − 1 to increase in concentration. Te high total favonoid and (2000–2500 cm ) was detected, implying the absence of total phenolic content of methanol fraction is evident that C ≡ C bond in the molecule, although transition metal − 1 the fraction contained hydroxyl groups that confers suf- carbonyl was observed at band of 2050 cm . Regarding the − 1 cient antioxidant activity against DPPH and NO radicals double bond region (1500–2000 cm ), aldehyde and ether- − 1 which is in accordance to the report made by Hassan et al. oxy compound were present at peaks of 1733 cm and − 1 . 1640 cm , respectively. In the fngerprint region − 1 Te study utilized the previous analytical techniques to (400–1500 cm ), carbonate ion was present at strong − 1 − 1 - identify diferent compounds present in the respective visible band of 1457 cm . At bands of 1364 cm , 1244 cm − 1 fractions on the basis of the nature of solvent as well as 1, and 1170 cm , aliphatic nitro compounds, aromatic nature of target compound. Te LC-MS investigation of ethers, and alcohol-hydroxy compound were present. A − 1 − 1 methanol fraction reveals the presence of thirteen com- strong visible band was observed at 1095 cm , 1025 cm , − 1 − 1 − 1 pounds and three compounds in ethyl acetate fraction, re- 984 cm , 827 cm , and 723 cm revealing cyclic ether, spectively, as shown in Tables 5 and 6. However, a favonoid ether-oxy compounds, phosphate ion, aromatic ring, and identifed as astilbin was present in the methanol fraction phenyl component in the molecule. 10 Journal of Analytical Methods in Chemistry Table 7: GC-MS profle of petroleum ether fraction of Ficus platyphylla stem bark ethanolic extract. Peak Molecular Retention Peak area Compound Quality number formula time (%) 1 n-Decanoic acid C H O 6.897 0.36 96 10 20 2 2 n-Hexadecanoic acid C H O 8.803 0.98 97 16 32 2 3 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester C H O 9.247 0.32 80 16 22 4 Benzenesulfonothioic acid, S-phenyl ester C H O S 9.500 0.81 95 12 10 2 2 5 Hexadecanoic acid, methyl ester C H O 11.359 0.37 94 17 34 2 6 Dibutyl phthalate C H O 11.546 1.18 97 16 22 4 7 n-Hexadecanoic acid C H O 12.368 14.29 99 16 22 2 8 9,12-Octadecadienoic acid (Z,Z)-methyl ester C H O 12.893 0.72 97 18 32 2 9 Trans-13-Octadecenoic acid, - methyl ester C H O 13.162 0.74 99 19 36 2 10 n-Hexadecanoic acid C H O 14.589 3.87 99 16 22 2 11 9,17-Octadecadienal, (Z) 14.787 0.49 95 12 trans-13-Octadecenoic acid, -methyl ester C H O 14.975 0.93 96 19 36 2 13 Trans-13-octadecenoic acid C H O 15.973 38.07 99 18 34 2 14 Octadecanoic acid C H O 16.384 4.81 99 18 36 2 15 Oleic acid C H O 17.110 2.16 98 18 34 2 16 1,4-benzenedicarboxylic acid, mono(1-methylethyl) ester C H O 22.959 0.33 30 11 12 4 17 cis-Vaccenic acid C H O 23.845 0.10 83 18 34 2 18 9-Octadecenal, (Z)- C H O 24.056 0.17 55 18 34 19 9-Octadecenal, (Z)- C H O 24.287 0.13 64 18 34 20 Cis,cis-7,10-Hexadecadienal C H O 25.152 1.53 59 16 28 21 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C H O 25.443 0.50 95 21 40 4 22 Octadec-9-enoic acid C H O 25.751 0.96 51 18 34 2 23 6-Octadecenoic acid, (Z)- C H O 26.309 1.99 66 18 34 2 24 9-Octadecenoic acid, (E)- C H O 27.117 5.05 58 20 38 2 25 Oleic acid C H O 27.230 1.04 53 18 34 2 26 Squalene C H 27.347 1.29 64 30 50 27 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C H O 27.955 5.20 92 21 38 4 28 9-Octadecenoic acid (Z)-2,3-dihydroxypropyl ester C H O 28.529 3.01 93 21 40 4 29 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester C H O 28.730 1.33 91 21 40 4 30 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C H O 29.339 2.99 94 21 38 4 Octadecanoic acid, 3-[(1-oxohexadecyl)oxy]-2-[(1-oxotetradecyl) 31 C H O 29.647 1.72 16 51 98 6 oxy]propyl ester 32 Tripalmitin C H O 30.132 0.70 14 51 98 6 33 n-Hexadecanoic acid, methyl(tetramethylene)silyl ester C H 0 Si 30.524 0.55 10 21 42 2 34 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C H O 35.207 1.33 90 21 40 4 Table 8: GC-MS profle of chloroform fraction of Ficus platyphylla stem bark ethanolic extract. Peak Molecular Retention Peak area Compound Quality number formula time (%) 1 Beta-sitosterol C H O 21.127 1.57 95 29 50 2 Gamma-sitosterol C H O 21.235 1.28 99 29 50 3 Oleic acid C H O 22.955 0.07 56 18 34 2 4 Z,Z-4,16-Octadecadien-1-ol acetate C H O 23.581 0.81 43 20 36 2 5 9,19-Cyclolanost-24-en-3-ol (3.beta.)- C H O 25.169 2.26 56 32 52 2 6 9-Octadecenoic acid (Z)-2-hydroxy-1-(hydroxymethyl)ethyl ester C H O 25.730 0.14 86 21 40 4 7 9-Octadecenoic acid (Z)-2,3-dihydroxypropyl ester C H O 27.242 2.18 93 21 40 4 8 3,4-Octadiene-2,2,7,7-tetramethyl C H 27.585 1.98 53 12 22 9 Olean-12-en-3-ol, acetate (3.beta.)- C H O 28.165 11.34 99 32 52 2 10 Lanosterol, TMS derivative C H OSi 28.342 3.60 52 33 58 11 12-Oleanen-3-yl acetate, (3.alpha.)- C H O 30.216 49.25 93 32 52 2 12 Lup-20(29)-en-3-ol, acetate (3.beta.)- C H O 30.333 21.65 99 30 50 13 Cholesta-8,24-dien-3-ol, (3.beta., 5.alpha.)-, TMS derivative C H O 30.724 0.57 90 29 48 Octadecanoic acid, 2-[(1-oxohexadecyl)oxy]-1-[[(1-oxohexadecyl) 14 C H O 32.601 0.38 22 53 102 6 oxy]methyl]ethyl ester 15 n-propyl-11-octadecenoate C H O 33.058 0.45 46 21 40 2 16 9-Octadecenoic acid, 2-(octadecyloxy)ethyl ester C H O 33.440 0.77 10 38 74 3 17 11,13-Dimethyl-12-tetradecen-1-ol acetate C H O 33.861 0.95 60 18 34 2 18 9-Octadecene-1-[3-(octadecyloxy)propoxy]-, (Z)- C39H O 34.285 0.74 81 78 2 Journal of Analytical Methods in Chemistry 11 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1) Figure 6: FTIR spectra of ethyl acetate fraction of ethanolic extract of Ficus platyphylla stem bark. 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1) Figure 7: FTIR spectra of methanol fraction of ethanolic extract of Ficus platyphylla stem bark. and has been established to possess certain biological is used as release agents, soap production, and cosmetics. functions ranging from antioxidant, antifungal, anti-car- Methyl esters are found in pheromones and essential oils and cinogen, and anticonvulsant properties . are also used as fragrance . Oleic acid and tripalmitin Te petroleum ether fraction analyzed via GC-MS have anticholesterolemic, anti-infammatory, antifungal, identifed thirty-four compounds as listed in Table 7 with antioxidative, and antibacterial properties . Another trans-13-octadecenoic acid being the main compound compound of importance identifed is Cis-vaccenic acid. Tis is an omega 7 fatty acid reported to decrease LDL- 38.07% and cis-vaccenic acid being the least compound 0.10%. One of the identifed phytochemicals, n-hex- cholesterol and improve insulin sensitivity . In addition, 9,12-octadecadienoic acid (Z,Z) methyl ester is a potent adecenoic acid has been reported in a previous study to possess an antioxidant, antibacterial, and antifungal prop- antioxidant that helps in prevention of prostate cancer erty [26, 27]. 9, 12-Octadecadienoic acid was reported to disease, Alzheimer disease, and cardiovascular diseases . possess anti-infammatory and antibacterial properties and Te chloroform fraction revealed eighteen compounds, also used in beauty and skin care products . Squalene has as shown in Table 8 with 12-Oleanen-3-yl acetate (3.alpha.) antioxidant, chemo-preventive activity against colon cancer, being the main compound 49.25% and beta-sitosterol being and anti-infammatory properties [26, 27]. To elaborate the least compound 0.07%. Beta-sitosterol acts in declining further, hexadecenoic acid is found mostly in plants, ani- the passage of cholesterol content in the blood vessels mals, or micro-organism as a form of saturated fatty acid. It through the inhibition of cholesterol absorption at the Transmittance Transmittance 12 Journal of Analytical Methods in Chemistry 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1) Figure 8: FTIR spectra of petroleum ether fraction of ethanolic extract of Ficus platyphylla stem bark. 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1) Figure 9: FTIR spectra of chloroform fraction of ethanolic extract of Ficus platyphylla stem bark. digestive track. It is also very essential in other body pro- bond, a phenyl, carbonyl, nitro, and peroxide components. cesses due to its anti-infammatory properties and improves Te chloroform fraction contained a long aliphatic chain kidney functions. Lanosterol is utilized to alleviate lens (methyl and methylene), a carbonyl, phenyl, ether-oxy and an opacity in age-related cortical cataract . Te compounds aromatic ring in the molecule. Te previous correlations were identifed in Table 7 have common biological activity which made in correspondence to the frequency range and func- include anti-infammatory, anti-bacterial, anti-fungal, anti- tional group assignment reported by Nandiyanto et al. . oxidant, anti-coronary, anti-acne, and anti-eczemic prop- erties . 7. Conclusion Te FTIR analysis of ethyl acetate fraction contains compound with a hydroxyl group, an aromatic ring, a long Te search for lead compounds from natural sources in the saturated aliphatic chain, nitro compounds, a double bond, management of several pathological conditions is endless. absence of triple bond, an aldehyde, ethers, and peroxide Te outcome of this study reveals the antioxidant capacity components. 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