Gas chromatography-mass spectrometry phytochemical studies of ethanol leaf extract of Annona muricata

 Corresponding author: Ene A.C Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Gas chromatography-mass spectrometry phytochemical studies of ethanol leaf extract of Annona muricata Ene Aloysius Chinedu 1, *, Opurum Christian Chibuzor 2, Iwu Irenus Chinonye 3, Chukwudoruo Chieme Sunday 1, Ibeh Raymond Chigozie 1 and Chinekeokwu Rufus Kelechukwu Christian 1 1 Department of Biochemistry, School of Biological Sciences, Federal University of Technology Owerri, Imo State, Nigeria. 2 Department of Microbiology, School of Biological Sciences, Federal University of Technology Owerri, Imo State, Nigeria. 3 Department of Chemistry, School of Physical Sciences, Federal University of Technology Owerri, Imo State, Nigeria. World Journal of Advanced Research and Reviews, 2025, 26(03), 2673-2679 Publication history: Received on 07 May 2025; revised on 25 June 2025; accepted on 27 June 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.3.2353 Abstract Folk medicine has taken an important place especially in developing countries where limited health services are available. However, the absence of scientific evaluation of medicinal plants may cause serious adverse effects. The phytochemical composition of the ethanolic extracts of leaves of Annona muricata was analyzed using gas chromatography -mass spectroscopy (GC-MS). The GC-MS analysis revealed thirty (30) constituents of which all were matched and identified. The major constituents were two compounds with percentage peak areas of 17.21% and 17.10%. Of the identified compounds, the outstanding in composition were 1,2-Benzisothiazol-3-amine, TBDMS Derivative, Propanamide, N-(4-methoxyphenyl)-2,2-dimethyl-1H-Indole with (peak area 17.21%), 6-Octadecenoic acid, (Z)-9Octadecenoic acid, (E)-6-Octadecenoic acid (peak area 17.10), 9-Octadecenoic acid (Z)-, 2-hydroxyethyl ester, Oleic Acid, 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester (peak area 15.54%). The current study suggests that ethanolic extracts of leaves of Annona muricata are a potent therapeutic agent and paves the way for the development of several treatment regimens based on compounds from this extract. Keywords: Annona muricata; Ethanolic Leaf Extracts; GC-MS Analysis; Phytochemicals 1. Introduction Plant use in treatment of diseases is as old as civilization (1,2) and complementary medicine is still a major part of habitual treatments of different maladies (2,3). Generally, complementary medicine has a long history of serving people all over the world (4,5). In recent times and due to historical, cultural, and other reasons, folk medicine has taken an important place especially in developing countries where limited health services are available. However, the absence of scientific evaluation of medicinal plants may cause serious adverse effects (3,6,7). Natural products are extremely an important source of medicinal agents. Although there are some new approaches to drug discovery, such as combinatorial chemistry and computer based molecular modeling design, none of them can replace the importance of natural products in drug discovery and development (8). Advancement in analytical technique has been extensively utilized for identification, structural determination and separation of bioactive compounds in a mixture by utilizing GCMS (9). Annona genus belongs to Annonaceae family classified under flowering plant. Species is muricata, common name: Soursop. World Journal of Advanced Research and Reviews, 2025, 26(03), 2673-2679 2674 Many non-natural, synthetic drugs cause severe side effects that were not acceptable except as treatments of last resort for terminal diseases such as cancer and that the metabolites discovered in medicinal plants may avoid the side effect of synthetic drugs, because they must accumulate within living cells (5). Annona muricata L commonly known as Soursop belongs to the family of Annonaceae and is the most tropical semi deciduous tree with the largest fruits of the Annona genus. It is a typical tropical tree with heart shaped edible fruits and widely distributed and native to SubSaharan Africa countries that lie within the tropics including Nigeria (7). The leaves are lanceolate with glossy and dark green in color and had been traditionally used to treat headaches, hypertension, cough, asthma and used as antispasmodic, sedative and nervine for heart condition, (10,11) as well as cancer. It is widely used for complementary treatment in many countries such as Amazonia, Barbados, Borneo, Brazil, Cook Islands, Curacao, Dominica, Guatemala, Guam, Guyana, Haiti, Jamaica, Madagascar, Malaysia, Peru, Suriname, Togo and West Indies, (12,13,14) as well as in Nigeria (7). It is hoped that traditional medicine will in future provide the cure to many tropical diseases that have defied orthodox prescriptions. Annona muricata leaves possess lots of secondary metabolite, vitamins and mineral with variable valuable biological activities. Hence, this informs the decision to carryout phytochemical screening of the leaf ethanol extract of Annona muricata using GCMS (9). 2. Materials and methods 2.1. Sample collection and authentication Fresh leaves of Annona muricata L. were collected from Ogidi town of Anambra State in Nigeria during the month of February 2025. The plant was identified and authenticated by Mr. Francis Iwunze from the Department of Forestry and Wildlife, School of Agricultural Technology, Federal University of Technology Owerri, Nigeria. A voucher specimen was deposited in the herbarium under the collection number FUTO/FWT/ERB/2025/71. 2.2. Samples preparation and Extraction The leaves of Annona muricata were washed with water and cut into small pieces, drying was done at room temperature under the shade for about thirty days, and the dried leaves were powdered by using a blender. 100 g of powdered leaves were extracted using 700 ml ethanol for three days by the plant tissue homogenization method as previously described (15). The extract was then concentrated using a rotary evaporator and stored in the refrigerator at 4oC until used. 2.3. Chemicals and reagents All chemicals and reagents were procured from certified suppliers and were of the highest analytical standard. 2.3.1. Gas Chromatography Mass Spectroscopy Gas chromatography mass spectroscopy (GC-MS), a hyphenated system which is a very compatible technique and the most commonly used technique for the identification and quantification purpose was used. The unknown organic compounds in the complex mixture can be determined by interpretation and also by matching the spectra with reference spectra (16). 2.3.2. Preparation of extract The ethanolic extract of the leaves was analyzed using Gas Chromatography Mass Spectroscopy for the identification of the phytochemical compounds present. A solvent blank analysis was first conducted using 1 μl of absolute ethanol. Then 1 μl of the reconstituted ethanolic extract solution was employed for GC-MS analysis as previously described with modifications (16,17) 2.3.3. Analysis GC-MS analysis was carried out on a GC system comprising a Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) instrument; Schimadzu GCMS-QP2010, employing the following conditions: Column Elite-1 fused silica capillary column (30×0.25 mm ID×1EM df, composed of 100% Dimethyl poly siloxane), operating in electron impact mode at 70 eV; helium (99.999%) as carrier gas at a constant flow of 1ml/ minute and a sample injection volume of 1 μl which was employed (split ratio of 10:1) injector temperature 250°C; ion-source temperature 280°C. The oven temperature was programmed from 110°C (isothermal for 2 minutes), with an increase of 10°C/minute, to 200°C, then 5°C/minute to 280°C, ending with a 9 minutes isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of World Journal of Advanced Research and Reviews, 2025, 26(03), 2673-2679 2675 0.5 s and fragments from 40 to 550 Da. Total run time was 30 min. The compounds were then identified from the GCMS peaks, using library data of the corresponding compounds. GC-MS was analyzed using electron impact ionization at 70 eV and data was evaluated using total ion count (TIC) for compound identification and quantification. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS library using NISP Search. The relative % amount of each component was calculated by comparing its average peak area to the total areas. Measurement of peak areas and data processing were carried out by Turbo-Mass-OCPTVS-Demo SPL software (17). 3. Results The results obtained from this study represented an important step towards the effective characterization of the secondary class metabolite compounds from this plant using GC-MS analysis. Preliminary qualitative phytochemical analysis of extracts revealed it to be rich in secondary class metabolite compounds of alkaloids, saponins, terpenoids, flavonoids, coumarins and lactones, anthraquinones, tannins, Cardiac glycosides, phenols and phytosterols. Each row in table 1 lists a unique bioactive component identified within the extract, along with its retention time during the analysis and the percentage it constitutes of the total extract. The results indicated that thirty (30) phytochemical constituents were identified from the leaf ethanol extract of Annona muricata (Table 1). Table 1 Phyto-components generated in the ethanolic leaves extract of Annona muricata by GC-MS analysis Peak # Retention time/ minutes % Composition by Area Bioactive Components Comment 1 10.193 0.43 Hexadecane Hexadecane 2,6,10-Trimethyltridecane Matched 2 13.024 0.10 9-Eicosene, 3-Eicosene, E-14-Hexadecenal Matched 3 13.171 0.34 1-Octadecanesulphonyl chloride 10-Methylnonadecane Tritetracontane Matched 4 14.710 0.54 Dibutyl phthalate Dibutyl phthalate 1,2-Benzenedicarboxylic acid, buty –loctyl ester Matched 5 15.771 0.45 1-Octadecene 1-Docosene 1-Nonadecene Matched 6 15.898 0.43 Sulfurous acid, butyl tetradecylester, Tetratetracontane Methoxyacetic acid, 2-tetradecyl ester Matched 7 18.289 0.34 1-Docosene 1-Hexadecanol, 2-methyl-9-Nonadecene Matched 8 18.402 0.26 Octadecanesulphonyl chloride, Carbonic acid, Tridecyl vinyl ester, Tetratetracontane Matched World Journal of Advanced Research and Reviews, 2025, 26(03), 2673-2679 2676 9 20.609 1.54 1-Docosene 1-Docosene Cyclotetracosane Matched 10 20.707 0.14 Docosane Sulfurous acid, butyl heptadecyl ester, Carbonic acid, eicosyl vinyl ester Matched 11 21.516 0.34 2-Piperidinone, N-[4-bromo-n-butyl,]- Oxirane, tridecylCyclohexane, 1,1'-(1-methyl-1,2ethanediyl) bisMatched 12 21.783 3.34 Bis(2-ethylhexyl) phthalate Bis(2-ethylhexyl) phthalate Bis(3-methylbutan-2-yl) phthalate Matched 13 22.164 0.29 2-Piperidinone, N- [4-bromo-n-butyl, 1-Heptadecene, Hexadecane, Matched 14 22.756 1.54 17-Pentatriacontene Octacosyl, trifluoroacetate Pentadecafluorooctanoic acid, octa decyl ester Matched 15 22.841 0.84 Docosane, 9-octylCarbonic acid, eicosyl vinyl ester, Tritetracontane Matched 16 24.857 0.43 Squalene Supraene, Squalene Matched 17 30.006 0.23 4-Cholesten-3-one semicarbazone, Hydrazinecarboxylic acid, 1,1-dimethylethyl ester, N-Acetoacetyl-deacetylcolchicine Matched 18 30.860 0.10 1,2-Benzisothiazol-3-amine, TBDMS derivative, Trichloroacetic acid, 2-tetradecyl ester 3-Trifluoroacetoxypentadecane Matched 19 30.934 0.43 1,2-Benzenediol, 3,5-bis(1,1-dimethylethyl)- 1,4-Benzenediol, 2,5-bis(1,1-dimethylethyl)- 2-(Acetoxymethyl)-3-(methoxycarbonyl)biphenylene Matched 20 31.034 0.11 Erucic acid, Heptadecanolide, Cyclopentadecanone Matched 21 31.052 0.03 Pentatriacontane, 13-docosenylidene-2-Pentadecanol, 2-Trifluoroacetoxypentadecane Matched 22 31.295 2.84 Propanamide, 2,2-dimethyl-N-(4-methylphenyl)- Propanamide, 2,2-dimethyl-N-(3-methylphenyl)- 2-(2-Butoxyethoxy) ethyl 2,2,3,3,3-pentafluoropropanoate Matched 23 31.335 2.45 Pyrrolidine, 1-methyl-3,2'-spiro-benzo-1,3-dioxolane-2,6-Ditert-butyl-4-methylphenol, O-heptafluorobutyryl, Pregn-16-en-20-one, 3-hydroxy-,.beta.,5.beta.)- Matched World Journal of Advanced Research and Reviews, 2025, 26(03), 2673-2679 2677 24 33.372 17.21 1,2-Benzisothiazol-3-amine, TBDMS Derivative, Propanamide, N-(4-methoxyphenyl)-2,2-dimethyl-1H-Indole, Matched 25 33.448 3.54 1,2-Benzisothiazol-3-amine, TBDMS derivative, Benzo[h]quinoline, 2,4-dimethyl-Cyclotrisiloxane, hexamethyl Matched 26 33.473 9.26 Acetamide, N-(4-fluorophenyl)-2,2, 2-trifluoro-1,2Benzisothiazol-3-amine, TBDMS derivative, 9-Octadecene, 1-[3-(octadecyloxy)propoxy]-(z) Matched 27 33.553 14.10 1,2-Benzisothiazol-3-amine, TBDMS derivative, 2-Ethylacridine, 9(10H)-anthracenone, 10,10-dimethyl Matched 28 34.901 2.83 Cyclotrisiloxane, hexamethyl-9-Borabicyclo [3.3.1] nonane, 9-[3-(dimethylamino)propyl]- Benzo[h]quinoline, 2,4-dimethyl Matched 29 35.384 17.10 6-Octadecenoic acid, (Z)- 9-Octadecenoic acid, (E)- 6-Octadecenoic acid Matched 30 36.755 15.54 9-Octadecenoic acid (Z)-, 2-hydroxyethyl ester, Oleic Acid, 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester Matched The peaks in the chromatogram were integrated and were compared with the database of spectrum of known components stored in the GC-MS NISP library. Phytochemical analysis by GC-MS analysis of the ethanolic extract of leaves of Annona muricata revealed the presence of different fatty acids, heterocyclic compounds, esters among others. Thirty peaks were generated. The detailed tabulations of GC-MS analysis of the extracts are given in Table 1. From the analysis, thirty compounds were elucidated in this study on Annona muricata, of which all compounds were effectively matched and identified. The leaf ethanolic extract of the plant generated thirty constituents, the major constituents were at peaks 24 (peak area 17.21%), peak 29 (peak area 17.10%), 6-Octadecenoic acid, (Z)-9-Octadecenoic acid, (E)-6-Octadecenoic acid, peak 30 (peak area 15.54%), 9-Octadecenoic acid (Z)-, 2-hydroxyethyl ester, Oleic Acid, 9-Octadecenoic acid (Z)-, 2,3dihydroxypropyl ester, Peak 27 (peak area 14.10%), 1,2-Benzisothiazol-3-amine, TBDMS derivative, 2-Ethylacridine, 9(10H)-anthracenone, 10,10-dimethyl, peak 26 (peak area 9.26%) Acetamide, N-(4-fluorophenyl)-2,2, 2-trifluoro-1,2Benzisothiazol-3-amine, TBDMS derivative, 9-Octadecene, 1-[3-(octadecyloxy)propoxy]-(z), peak 25 (peak area 3.54%) 1,2-Benzisothiazol-3-amine, TBDMS derivative, Benzo[h]quinoline, 2,4-dimethyl-Cyclotrisiloxane, hexamethyl, peak 12 (peak area 3.34%) Bis(2-ethylhexyl) phthalate, Bis(2-ethylhexyl) phthalate, Bis(3-methylbutan-2-yl) phthalate, peak 22 (peak area 2.84%) Propanamide, 2,2-dimethyl-N-(4-methylphenyl)-Propanamide, 2,2-dimethyl-N-(3methylphenyl)- 2-(2-Butoxyethoxy)ethyl 2,2,3,3,3-pentafluoropropanoate, peak 23 (peak area 2.45 %) Pyrrolidine, 1methyl-3,2'-spiro-benzo-1,3-dioxolane-2,6-Di-tert-butyl-4-methylphenol, O-heptafluorobutyryl,Pregn-16-en-20-one, while the rest had less than 2% composition by peak area. 4. Discussion The presence of various secondary class metabolites identified puts these results in line with earlier studies that were carried out on the ethanolic seeds extract of Annona muricata, and the phytochemical tests showed that ethanol soursop seeds extract contains secondary metabolites compounds group of saponins, alkaloids and triterpenoids, flavonoids, anthraquinones, tannins, and cardiac glycosides, which they noted that they are defense chemical compounds of plants produced in the plant tissue (17,18). The plant could thus be used for the management of various healthy conditions associated with the metabolites screened. Using GC-MS Analysis, 30 compounds have been elucidated for the first time in our study on Annona muricata, of which all the compounds were effectively matched and identified. 1,2-Benzenedicarboxylic acid, butyl octyl ester is a plasticizer compound with antimicrobial, antifouling, antioxidant and hypo-cholesterolemic activities. (19) 3,7,11,15-Tetramethyl-2-hexadecen-1-ol is recorded to have antituberculosis, insecticidal, anti-inflammatory, antioxidant and antimicrobial activities. n-Hexadecanoic acid on the other World Journal of Advanced Research and Reviews, 2025, 26(03), 2673-2679 2678 hand which is commonly known as Palmitic acid has nematicide, pesticide, lubricant, anti-androgenic, flavor, hemolytic 5-alpha reductase inhibitor, antioxidant and hypo-cholesterolemic properties. (17). Hexadecanoic acid, ethyl ester is a fatty acid ester with nematicide, pesticide, lubricant, anti-androgenic, flavor, and has hemolytic 5-alpha reductase inhibitor properties. (20,21) Phytol and phthalate are diterpenes with antimicrobial, anticancer, anti-inflammatory, anti-diuretic, immunestimulatory and anti-diabetic properties. 9,12-Octadecadienoic acid, ethyl ester is a linoleic acid which has hypocholesterolemic, 5-alpha reductase inhibitor, antihistaminic, insectifuge, anti-eczemic, and anti-acne properties. Finally, 1,2-Benzenedicarboxylic acid, diisooctyl ester is a plasticizer compound with antimicrobial and antifouling properties. (16,20,21) It is worth noting that of the major constituents identified in our extract are the compounds with the highest composition at peaks 24 (peak area 17.21%) and Peak 29 (peak area 17.10%). These two could be very novel compounds that need to be analyzed further in order to elucidate their nature. The same applies to the remaining 28 compounds which have lower concentrations. 5. Conclusion GC-MS analysis of the ethanolic extract of leaves of Annona muricata revealed the presence of different fatty acids, heterocyclic compounds, esters among others. This confirms the results on presence of the various secondary metabolite compounds detected by the qualitative procedures. These mass spectra are fingerprint of the compound which can be identified from the data library. 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