Phytochemical composition and GC-MS analysis of Hymenodictyon orixense Mabb. leaves

 Corresponding author: Vartika Jain 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. Phytochemical composition and GC-MS analysis of Hymenodictyon orixense Mabb. leaves Anju Kumari 1 and Vartika Jain 2, * 1 Department of Botany, S. K. Government Girls’ College, Sikar-332001, Rajasthan, India. 2 Department of Botany, Government Meera Girls’ College, Udaipur-313001, Rajasthan, India. GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 Publication history: Received on 06 August 2025; revised on 13 September 2025; accepted on 15 September 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.32.3.0357 Abstract Hymenodictyon orixense Mabb. (Rubiaceae); commonly known as Bridal Couch Tree, is used to treat ulcer, fever, inflammation, malaria, menstrual disorders and other ailments by ethnic communities of India. The present study aimed to document phytocompounds present in methanolic leaf extract using qualitative phytochemical screening and Gas Chromatography-Mass Spectrometry (GC-MS) analysis for the first time. Phytochemical screening revealed the presence of protein, carbohydrate, tannin, phlobatannin, alkaloid, cardiac glycoside, phytosterol, terpenoid, saponin, phenol and flavonoid. In quantitative tests, the total phenolic and flavonoid contents were found to be 62.57±4.28 mg GAE/g and 14.89±0.95 mg QE/g, respectively. GC-MS approach exhibited presence of 46 phytocompounds as determined through fragmentation pattern, molecular weight, molecular formula, and phytocomponent structure. The major compounds identified were, 1,3,4,5-tetrahydroxy-cyclohexanecarboxylic acid ( 14.34% ), Squalene ( 14.25% ), βsitosterol ( 11.37%) , (z)-9-octadecenamide ( 9.25% ), Phytol ( 6.92% ) and Ergost-5-en-3-ol ( 4.59% ) etc. The results exposed the rich phytochemical profile of H. orixense leaves suggesting its further in vitro, in vivo and in silico exploration for assessment of pharmacological activities and structural characterization of bioactive molecules. Keywords: Flavanoids; Quinic Acid; Rubiaceae; Squalene; Terpenoids; Vitamin E 1. Introduction Medicinal plants have been fundamental to traditional healthcare systems for ages and remain crucial in modern pharmacotherapy [1]. They play a major role in drug development research due to their medicinal potential and as scientific support for ethnobotanical assertions. Despite the breakthroughs in synthetic drug discovery, natural products are preferred due to their therapeutic chemical structures and biological properties. Their growing acceptance is attributed to established roles in traditional medicine and benefits including safety, cost-effectiveness, and versatility [2]. Phytochemical analysis is fundamental to pharmacognostic investigations, focusing on the separation, identification, and quantification of bioactive molecules in medicinal plants. These substances, termed as secondary metabolites, exhibit diverse pharmacological actions and are categorised into several categories, for example, alkaloids, flavonoids, terpenoids, glycosides, phenolic compounds, essential oils etc. [3]. Phytochemicals have also been demonstrated to exhibit various biological activities, for example, antioxidant, antidiabetic, anti-inflammatory, antimicrobial, anticancer etc. [4]. The qualitative and quantitative investigation of these phyto-constituents provides insights that are crucial for substantiating traditional knowledge in a scientific manner and overall valuable for novel drug discovery [5]. GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 157 Gas Chromatography-Mass Spectrometry (GC-MS) is a very effective analytical technique utilized for the identification and characterization of volatile and semi-volatile chemicals in complex plant matrices. Gas chromatography separates mixtures into distinct components utilizing a temperature-regulated capillary column [6,7]. In addition to volatile compounds, GC-MS is also utilized to elucidate structure of a wide range of compounds, such as steroids, glycosides, essential oils, flavonoids, phenols, alkaloids, saponins, and their derivatives [8]. The preliminary phytochemical screening with GC-MS profiling offers a more thorough comprehension of the bioactive compounds present in the plant extracts, facilitating the discovery of essential therapeutic phyto-constituents [9]. Usually, methanolic extracts are preferred in these investigations as methanol effectively extracts a broad range of polar and somewhat non-polar phytochemicals [10]. Hymenodictyon orixense Mabb. (Figure 1.); a member of the Rubiaceae family, is called as Bridal Couch Tree, Bhorsal, Kadambu, Bhurkund, Kusan, Bhorkud, Kukurkat, Diddi Mara, Kala Bachnag, etc. in different languages [11]. It is used for treatment of skin ailments, inflammation, fever, atrophy, ulcerous wounds, sores, cholera, malaria, liver complaints, body swelling, diarrhoea, gout, menstrual disorders, snake bite, smallpox etc. by ethnic communities of India [12,13]. It has also revealed presence of various phytochemicals and several pharmacological activities, such as antioxidant, antimicrobial, antimalarial, antipyretic, anti-atherothrombotic, central nervous system depressant, cytotoxic, and acetylcholinesterase inhibition [11,14]. Despite the medicinal value, there exists a paucity of scientific studies regarding the phytochemical composition of H. orixense leaves, especially through utilising advanced techniques such as GC-MS. Therefore, the present research work has been envisaged to determine its phyto-constituents profile that will be helpful to validate the traditional medicinal applications of the plant as well as provide a basis for subsequent phytopharmaceutical researches. 2. Materials and Methods 2.1. Plant collection and authentication The leaves of H. orixense were collected from Amberi, Udaipur, Rajasthan, India in June 2023. The voucher specimen was prepared (Field no. 08) and authentication of the plant was carried out at Arid Zone Regional Centre, Botanical Survey of India (BSI), Jodhpur (No. BSI/AZRC/l.12012/2023-24/Tech. (Pl.Id.)/317 dated 03.08.2023). Figure 1 Hymenodictyon orixense Mabb 2.2. Preparation of plant extract The leaves were air dried under shade then powdered and kept in an air-tight container. The extraction was done using the cold extraction method. Ten-gram leaves powder was dissolved in 100 ml of methanol, and was kept on a shaker at 30°C at 90 rpm for 72 hours. The extract was filtered and kept for drying in pre-weighed petri dish at room temperature till it gained constant weight, labelled, and stored until use. The percent yield was calculated as follows: Extraction yield (%) = Weight of crude extract/ Weight of dried powder × 100 GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 158 2.3. Preliminary qualitative phytochemical screening Preliminary phytochemical screening of methanolic leaf extract was conducted to determine the presence or absence of various phyto-constituents such as carbohydrates, proteins, alkaloids, flavonoids, terpenoids, tannins, phytosterols, cardiac glycosides and saponins using standard methods with slight modifications [15]. 2.4. Assessment of Total Phenolic Content (TPC) The total phenolic content in plant extract was quantified using a spectrophotometric technique. The Folin-Ciocalteu method was employed to ascertain the total phenol content. The reaction mixture comprises 1 ml of extract and 9 ml of distilled water, contained within a 25 ml volumetric flask. 1 ml of Folin-Ciocalteu phenol reagent was added to the mixture and thoroughly agitated. After 5 minutes, 10 ml of a 7% sodium carbonate (Na2CO3) solution was added to the mixture and the volume was made up to 25 ml. A series of standard solutions of gallic acid (50, 100, 150, 200, 250, 300, 350, 400, 450 μg/ml) were also made in the same manner. After incubation for 90 minutes at an ambient temperature, the absorbance of test and standard solutions was measured against the blank at 550 nm using a UV/Visible spectrophotometer. The total phenolic content was quantified as mg of GAE/g of extract [16]. 2.5. Assessment of Total Flavonoid Content (TFC) Total flavonoid content was estimated using a colorimetric assay with aluminium chloride. The reaction mixture contains 1 ml sample and 4 ml of distilled water in a 10 ml flask. To the mixture, 0.3 mL of 5% sodium nitrite was mixed and 5 minutes later, 0.3 ml of 10 % aluminium chloride was added. After five minutes, 2 ml of 1M Sodium hydroxide (NaOH) was mixed and made up to 10 ml using distilled water. A set of standard solutions (positive control) of quercetin (50, 100, 150, 200 and 250 μg/ml) were prepared similarly as explained earlier. The absorbance for sample and the standard was taken against the blank reagent at 510 nm using an UV/Visible spectrophotometer. The total flavonoid content was demonstrated as mg of QE/g of extract [16]. 2.6. GC-MS Analysis The GC-MS analysis was performed at the Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, Delhi. GC-MS analysis was conducted using a GC-MS-QP-2010 Plus Ultra. The GC/MS equipment utilised a Rxi-5 SIL MS column of 30 m × 0.25 mm × 0.25 µm in film thickness, consisting of 5% diphenyl and 95% dimethyl polysiloxane, and operated in electron impact mode at 70 eV. The carrier gas utilised was helium, maintaining a constant flow rate of 1.21 ml/min, with an injection volume of 1 µl and a split ratio of 10:1. The oven temperature was originally set to 60 °C for 2 minutes, and thereafter scheduled to rise to 300 °C at a rate of 10 °C/min for 19 minutes. The operational conditions for the MS were as follows: Interface Temperature: 270 °C, Ion Source Temperature: 220 °C, Solvent Cut Time: 2.50 minutes, Scan Speed: 3333, Mass Scan Range (m/z): 40-600, Threshold: 1000. GC-MS was examined via electron impact ionization at 70 eV, and the data was assessed using Total Ion Count (TIC) for the identification and quantification of compounds. The relative percentage of each component in the GC-MS spectrum was determined by comparing the average peak area of individual compounds to their total areas. The identification of compounds was determined by comparing their retention times and mass spectra with reference compounds from the NIST/Wiley spectral database. The biological activities of the compounds identified through GC-MS are also given in Table 3 after thorough search of online scientific databases such as Google Scholar, Science Direct, ResearchGate, Wiley etc. 3. Results 3.1. Extraction yield In the present study, the extraction yield obtained for methanolic leaves extract of H. orixense was 9.66%. The extract exhibited a dark green colour. 3.2. Preliminary phytochemical screening A qualitative analysis of the methanolic extract of H. orixense leaves was conducted to detect the presence of various phytochemicals (Table 1). The result revealed the presence of primary and secondary metabolites, including carbohydrates, proteins, saponins, tannins, alkaloids, cardiac glycosides, phytosterols, phlobatannins, phenols, flavonoids and terpenoids. GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 159 Table 1 Preliminary qualitative phytochemical screening of methanolic leaves extract of H. orixense Phytochemical test and method Result Carbohydrate (Fehling’s test) + Protein (Ninhydrin test) + Tannin (Gelatin solution+ NaCl test) + Alkaloid (Mayer’s test) + Cardiac glycoside (NaOH test) + Saponin (Honey comb test) + Phytosterol (Libermann-Burchard’s test) + Phlobatannin (HCl test) + Phenol (FeCl3 test) + Flavonoid (Lead acetate test) + Terpenoid test (Salkowski test) + + = present 3.3. Total Phenolic Content (TPC) Total phenolic content was determined utilizing the Folin–Ciocalteu method. The result was obtained from a calibration curve (y = 0.0007x + 0.1072, R2 = 0.9893) of the standard compound gallic acid (50–450 µg/ml) as shown in Figure 2. The concentration of total phenol is expressed as mg/g gallic acid equivalent (GAE). The total phenolic content was found to be 62.57 mg GA/g in the methanolic leaves extract of H. orixense (Table 2). Figure 2 Standard curve of Gallic acid at 550 nm for calculating Total Phenolic Content 3.4. Total Flavonoid Content (TFC) The flavonoid concentration was quantified as quercetin equivalents (mg of Q/g of extract). The concentration of flavonoids was derived from a standard calibration curve (y = 0.0016x + 0.0955, R2 = 0.9982) of quercetin (50–250 µg/mL) as shown in Figure 3. In the methanol extract, TFC was found to be 14.89 mg QE/g (Table 2). GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 160 Figure 3 Standard curve of Quercetin at 510 nm for calculating Total Flavonoid Content Table 2 Total phenolic and flavonoid contents in the methanolic leaf extract of H. orixense Total phenolic content (mg of GAE/g of extract) 62.57 ± 4.28 Total flavonoid content (mg of QE/g of extract) 14.89 ± 0.95 Results are expressed in terms of gallic acid equivalent (mg of GAE/g of extract) and quercetin equivalent (mg of QE/g of extract), respectively as mean ± standard deviation. 3.5. GC-MS Analysis GC-MS is a method that integrates the separation of phytochemicals using gas chromatography with their detection via mass spectrometry [17]. The methanolic extract of H. orixense leaves was analysed using GC-MS, revealing 50 peaks in the chromatogram (Figure 4). A total of 46 compounds were identified, among which 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid (14.34%), squalene (14.25%), β-sitosterol (11.37%), (z)-9-octadecenamide (9.25%), phytol (6.92%), and ergost-5-en-3-ol (4.59%) were the most abundant (Table 3). Figure 4 GC-MS Chromatogram of methanolic leaf extract of H. orixense GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 161 Table 3 Bioactive compounds identified in methanolic extract of H. orixense leaves using GC-MS technique Peak R. Time Area% Name Molecular formula Structure Mol. Weight (g/mol) Nature of compound Biological activities References 1. 5.113 1.88 2-Hydroxycyclopent-2en-1-one C5H6O2 98 Cyclic hydroxy ketone NR - 2. 5.479 0.16 3-Methyl-1-heptanol C8H18O 130 Alcohol NR - 3. 5.700 0.26 1-(2-Methyl-1-propenyl) pyrrolidine C8H15N 125 Pyrrolidine alkaloid derivative- (Specifically heterocyclic amine) NR - 4. 5.943 1.33 2,4-Dihydroxy-2,5dimethyl-3(2H)-furan-3one C6H8O4 144 Furanone derivative NR - 5. 6.055 0.98 2,2-Dimethyldecane C12H26 170 Hydrocarbon (Alkane) NR - GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 162 6. 8.666 1.63 3,5-Dihydroxy-6-methyl2,3-dihydro-4H-pyran4-one C6H8O4 144 Oxygenated heterocyclic compound – Pyranone derivative Anti-diabetic, Antioxidant, Antimicrobial, Antiinflammatory [6,18-20] 7. 10.106 0.21 1Dimethyl(isopropyl)silyl oxypropane C8H20OSi 160 Silyl ether NR - 8. 10.771 0.59 Undecan-2-one C11H22O 170 Ketone (Fatty acid derivative/Al iphatic ketone) Antioxidant, Antimicrobial [21,22] 9. 12.125 0.15 9-Methyl-1-undecene C12H24 168 Hydrocarbon (unsaturated alkene) NR - 10. 12.282 0.15 Bis-2-ethylhexylamine C16H35N 241 Alkyl amine NR - 11. 12.524 0.31 1Pyrrolidinocyclohexene C10H17N 151 Alkaloid (cyclic amine) Cytotoxic, Antimicrobial [23] 12. 12.932 0.33 Undecylcyclohexane C17H34 238 Hydrocarbon (Alkane) NR - GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 163 13. 13.956 0.31 Hydrazinecarboxylic acid, butylidene-, methyl ester C6H12N2O2 144 hydrazone ester / Hydrazine derivative NR - 14. 14.098 0.34 4,4,7a-Trimethyl5,6,7,7a-tetrahydro-1benzofuran-2(4H)-one (Dihydroactinidiolide) C11H16O2 180 Benzofurano ne (lactone) derivative Antioxidant, Acetylcholineste rase inhibition, Neuroprotective , Anticancer, Antibacterial [24] 15. 14.656 0.57 Hexadecanol (n-Cetyl alcohol) C16H34O 242 Fatty alcohol (Fatty acid derivative) Antimicrobial, Antiinflammatory [25] 16. 15.256 14.34 Quinic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid) C7H12O6 192 Phenolic acid derivative / Cyclitol derivative Antioxidant, Antidiabetic, Antibacterial, Antimicrobial, Antinociceptive, Anticancer, Antiviral, Analgesic, [26] GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 164 17. 16.067 2.85 MOME INOSITOL (3-O-Methyl-D-glucose) C7H14O6 194 Monosacchar ide derivative Antiinflammatory, Antitumor [27] 18. 16.883 1.52 6-Hydroxy-4,4,7atrimethyl-5,6,7,7atetrahydrobenzofuran2(4H)-one C11H16O3 196 Monoterpen e lactone Antiinflammatory [28] 19. 17.033 0.49 Pluchidiol C13H20 O2 - 208 Triterpene Antiinflammatory, Antioxidant [29] 20. 17.384 3.38 Neophytadiene (7,11,15-Trimethyl-3methylenehexadec-1ene) C20H38 278 Terpenoid (Diterpenoid ) Antioxidant, Antipyretic, Anti-microbial, Antiinflammatory, Vermifugic, Analgesic, Antibacterial [30,31] 21. 17.442 0.48 6,10,14-Trimethyl-2pentadecanone (Hexahydrofarnesyl acetone) C18H36O 268 Terpenoid (Acyclic Sesquiterpen oid Ketone) Antiinflammatory, Antimicrobial, Cytotoxic [32] 22. 17.636 0.41 Neophytadiene C20H38 278 Terpenoid As reported in S. No. 20 As reported in S. No. 20 23. 17.835 1.05 Neophytadiene C20H38 278 Terpenoid As reported in S. No. 20 As reported in S. No. 20 24. 18.288 1.15 Hexadecanoic acid, methyl ester C17H34O2 270 Fatty acid ester Antioxidant, [6,27,33,34] GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 171 Table 5 Distribution of phytochemicals analyzed through GC-MS S. No. Phytochemical group Total % Area 1. Terpenes and terpenoid derivatives 32.26 2. Fatty acid derivatives 18.69 3. Phenolic compounds and their derivatives 18.39 4. Phytosterols 17.21 5. Hydrocarbons 3.44 6. Miscellaneous compounds 10.01 Figure 4 Composition of phytocompounds in methanolic leaves extract of H. orixense after GC-MS analysis 4. Discussion Herbal medicines embody a combination of historic traditional knowledge and modern pharmacology, reflecting centuries of empirical learning from various civilizations [66]. However, a comprehensive scientific investigation of phytochemical composition of plants is necessary to assess their therapeutic efficacy. Bioactive molecules obtained from plants, play an important role in modern medicine owing to their varied therapeutic potential [67,68]. The qualitative screening of methanolic extract of H. orixense leaves exhibited a variety of phytochemicals, comprising carbohydrates, proteins, saponins, tannins, alkaloids, cardiac glycosides, phytosterols, terpenoids, phlobatannins, phenols, and flavonoids. The presence of these valuable secondary metabolites suggests that the plant holds significant potential for treating a range of human diseases and could serve as a promising source for the development of new therapeutic drugs. The constituents in the methanolic extract of H. orixense are categorised into major phytochemical groups based on their structural class as analyzed through the GC-MS data (Table 3 & 4, Figure 5). The GC-MS spectrum revealed 50 peaks, corresponding to 46 phytocompounds (Figure 4). Among these compounds, many possess pharmaceutical significance, with several earlier studies documenting their therapeutic potential, for example, antioxidant, antidiabetic, anticancer, antimicrobial, anti-inflammatory and other activities (Table 3). GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 172 Phenolic compounds and their derivatives comprised 18.39% of the total area, mostly dominated by quinic acid (14.34%), in addition to tocopherols and phenolic esters (Figure 5). The quantitative assessment of total phenolic content and total flavonoid content yielded values of 62.57 ± 4.28 mg GAE/g and 14.89 ± 0.95 mg QE/g, respectively (Table 2), reflecting a significant abundance of antioxidant compounds in the extract [69]. Flavonoids are among the most notable bioactive groups identified, distinguished by their broad pharmacological properties. These chemicals are recognised for their significant antioxidant, anti-carcinogenic and anti-inflammatory properties [70]. Their capacity to regulate essential cellular enzymes and signalling pathways renders them vital in the prevention and control of chronic diseases, including diabetes, cardiovascular disorders, and neurodegenerative problems [71]. Likewise, phenolic compounds have shown to enhance the antioxidant capacity of plant extracts and demonstrated anti-aging, antibacterial, anticancer, anti-diabetic, anti-inflammatory and antiviral effects [72,73]. The GC-MS profile revealed the presence of two tocopherol isomers, α-tocopherol and γ-tocopherol, which are effective phenolic antioxidants. γ-Tocopherol exhibits potent antioxidant, anti-inflammatory, and anticancer activities by neutralising reactive nitrogen species and regulating COX-2 and TNF-α pathways [50]. Similarly, α-tocopherol has also demonstrated antioxidant, anti-diabetic, anticancer, antibacterial, anti-inflammatory, antitumor, and anti-aging properties [33,53]. Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate is a sterically hindered phenolic compound that was previously isolated from Alcaligenes faecalis MT332429, demonstrates significant antifungal activity [65]. It is also extensively utilized in industrial applications due to its antioxidant properties [64]. Quinic acid is another powerful antioxidant linked to antidiabetic, antibacterial, and antiviral properties [26]. Tannins, prevalent in numerous medicinal plants, enhance gastrointestinal health and exhibit significant antioxidant, antimicrobial, anti-mutagenic, anti-inflammatory, anticancer, antidiabetic, cardioprotective, hypolipidemic and anthelmintic properties [74,75]. Terpenes and terpenoid derivatives constituted the predominant class, with 32.26% of the overall makeup (Table 4). This dominance was mostly attributable to elevated concentrations of squalene (14.25%), phytol (6.92%), neophytadiene, triterpenoid epoxides, and 6,10,14-trimethyl-2-pentadecanone (hexahydrofarnesyl acetone). Phytol (3,7,11,15-Tetramethyl-2-hexadecen-1-ol), a diterpenoid alcohol, is recognised for its diverse biological activities, including antioxidant, antidiabetic, anticancer, anti-inflammatory, antidiuretic, and antimicrobial effects [6]. Its derivative, phytyl tetradecanoate, similarly exhibits antioxidant, anticancer, anti-inflammatory, and antimicrobial properties and serves as precursors in the synthesis of vitamins E and K1 [62]. The occurrence of neophytadiene, a diterpene detected in methanolic extract of Abutilon pannosum leaf and ethanolic extract of Glochidion ellipticum leaf, indicates its potential functions in antioxidant, antimicrobial, antipyretic, analgesic, and anti-inflammatory activities, as well as its application in alleviating headache, rheumatism, and certain dermatological conditions [30,31]. The ethanolic extract of Polygonum chinense previously demonstrated that squalene, a triterpene, possesses anti-cancer, antioxidant, anti-tumor, chemo-preventive, pesticidal and sunscreen effects [47]. Squalene has also shown to possess anti-diabetic, anti-aging, analgesic, anti-inflammatory, antimicrobial, antileukemic, hepatoprotective, hypocholesterolemic activities [34,48]. A sesquiterpene, hexahydrofarnesyl acetone (6,10,14-trimethyl-2-pentadecanone), earlier identified in essential oil of Albizia zygia, essential oil of Lindera nacusu leaves and from essential oil of Graptophyllum pictum leaves, is distinguished by its antibacterial, anti-inflammatory, antioxidant and anticancer activities [32,76,77]. Likewise, 6hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one, a monoterpene lactone derived from the ethanolic extract of Sargassum horneri, also demonstrated anti-inflammatory efficacy by mitigating LPS-induced inflammation via the inhibition of pro-inflammatory mediators and the modulation of NF-κB and MAPK signalling pathways [28]. On the other hand, fatty acid derivatives constituted the second largest group with 18.69%, comprising fatty acid esters, alcohols, and amides (Table 3 & 4). Notably, z-9-octadecenamide (9.25%) a fatty acid amide, was the predominant compound in this group, which has exhibited significant antibacterial and anti-inflammatory properties [44] suggesting its potential role in the therapeutic efficacy of the extract. Palmitic acid methyl ester is recognised for its antioxidant [6,27,33], anti-diabetic, nematicidal, hypocholesterolemic [27], and antibacterial characteristics [6,34]. Methyl stearate has demonstrated antibacterial [34,40], antiproliferative, cytotoxic, and anti-diarrheal properties [37]. Linolenic acid methyl ester had also shown various pharmacological activities such as antibacterial, anti-inflammatory, hypocholesterolemic, nematicidal, anticancer, and hepatoprotective effects [20]. Another compound observed in the methanolic extract was hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (Glycerol 2-palmitate), which is a fatty ester well-known for its antioxidant, nematicide, hypocholesterolemic, and antiandrogenic activities [43]. GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 173 Phytosterols (17.21%) identified in the extract (Table 3 & 4) have structural similarities to cholesterol, facilitating cholesterol reduction and enhancing lipid metabolism. These chemicals exhibit anti-inflammatory, antioxidant, and anti-cancer properties, and are associated with a decreased risk of cardiovascular illnesses and Alzheimer’s disease [78]. For example, β-sitosterol, stigmasterol, and campesterol (ergost-5-en-3-ol) were detected in substantial amounts in leaves of H. orixense (Table 2). Previous studies have demonstrated that β-sitosterol exhibits various activities, such as antioxidant, anti-diabetic, anti-inflammatory, antimicrobial, anti-fertility, anti-obesity and anticancer [27,40,61]. The significant relative abundance of β-sitosterol in this study (11.37%) indicates a substantial contribution to the extract's bioactivity. Stigmasterol is also reported for its diverse activities, including cholesterol-lowering [56], anti-diabetic, neuroprotective, antiparasitic, immunomodulatory, antimicrobial, antioxidant, anticancer, anti-inflammatory, and antiosteoarthritis properties [57]. It has also demonstrated disruption in carcinogenic signalling pathways, including Akt/mTOR and JAK/STAT, and inhibition of angiogenesis via suppression of VEGFR-2 [57]. Campesterol has shown to diminish cholesterol absorption and also possesses anticancer and antioxidant properties [43,54,55]. Saponins, a significant category included in the extract, are recognised for their diverse biological actions. They possess immunostimulatory, hypocholesterolemic, anticancer, antibacterial, antiviral, antifungal, and anti-inflammatory activities [79]. Moreover, saponins have also demonstrated antihypertensive, hypoglycemic, and antioxidant properties, highlighting their potential in the management of metabolic syndromes and associated illnesses [80]. Cardiac glycosides, traditionally employed in the treatment of heart failure, have also been linked to antibacterial, antiinflammatory, anticancer, and neuroprotective properties [81]. Aliphatic hydrocarbons represented 3.44% (Fig. 5), primarily comprising long-chain alkanes such as heneicosane and branched alkanes such 2-methylhexacosane, which may serve in plant defense or as pheromone analogues [51] whereas heneicosane has shown antimicrobial properties [43]. 4,4,7a-Trimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one (Dihydroactinidiolide), derived from β-ionone, demonstrates significant multi-target efficacy against Alzheimer’s disease, characterized by robust acetylcholinesterase inhibition (IC₅₀ = 34.03 nM), antioxidant properties, metalchelating abilities, and anti-amyloid aggregation effects. It demonstrates remarkable neuroprotective potential without cytotoxicity, reinforcing its promise as a principal drug for Alzheimer's disease [24]. The miscellaneous compounds, comprising 10.01%, covered monosaccharide derivatives such as MOME inositol (2.85%), alkaloids and their derivatives (1.05%), lactones, non-alkaloidal nitrogen heterocyclic, pyranones, silyl ethers, and other minor compounds. Although individually present in small quantities, these compounds may produce specific pharmacological effects or function synergistically with the primary components. For example, alkaloids as nitrogenous compounds exhibit pharmacological versatility demonstrating analgesic, antihypertensive, anticancer, antibacterial, and anti-hyperglycemic properties. Additionally, several alkaloids demonstrate psychotropic and neuro-protective properties, rendering them potential candidates for the management of inflammatory and neurodegenerative disorders [82,83]. The alkaloids identified through GC-MS technique in the present study were, 1-(2-methyl-1-propenyl) pyrrolidine, 1-pyrrolidinocyclohexene, and 2,3-bis(1-methylallyl) pyrrolidine. The pyrrolidine nucleus is recognized as a favoured scaffold in drug discovery owing to its antioxidant, antibacterial, anticancer, and anti-inflammatory activities [84]. Although any specific biological activities of these individual alkaloids have not been demonstrated yet, but their structural characteristics imply therapeutic significance. Interestingly, several phytochemicals detected in the current study have been previously reported in some other plant species of Rubiaceae family, though their relative abundance was variable. The presence of 7.90% heneicosane was reported in the leaves of Morinda citrifolia [85]. Haruna identified squalene at 15.13% [86], whereas Danial et al. documented elevated squalene levels at 41.50%, in addition to phytol (13.06%), neophytadiene (10.06%), α-tocopherol (5.94%), stigmasterol (2.74%) and 9-octadecenamide (1.03%) in M. citrifolia [87]. Jeon et al. identified quinic acid (7.87%), 9-octadecenamide (3.84%), phytol (2.67%), neophytadiene (2.38%), stigmasterol (1.74%), γ-tocopherol (1.01%) and palmitic acid methyl ester (0.48%) in Arabica coffee leaves [88]. Likewise, 3,5-dihydroxy-6-methyl-2,3dihydro-4H-pyran-4-one (29.87%), squalene (14.58%), α-tocopherol (5.78%) and phytol (5.66%) were reported from leaves of Spermadictyon suaveolens [89]. In the present study, methanolic extract of H. orixense exhibited several of these compounds, but with varying concentrations. Quinic acid was present in higher concentration 14.34% in comparison to 7.87% in Arabica coffee. Squalene was found at 14.25%, nearly aligning with the 14.58% as documented for S. suaveolens; however, far lower than the remarkably high concentration of 41.50% in M. citrifolia. The fatty acid amide 9-octadecenamide constituted 9.25%, far above the 1.03% found in M. citrifolia and 3.84% in Arabica coffee, suggesting it may serve as a chemotaxonomic identifier for H. orixense. The concentration of phytol was higher (6.92%) than that found in Arabica GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 174 coffee (2.67%) and S. suaveolens (5.66%) but lower to M. citrifolia (13.06%). Similarly, Neophytadiene (3.38%) exceeded that of Arabica coffee (2.38%) but was lower than M. citrifolia (10.06%). α-Tocopherol was measured at 1.88%, which is lower than that found in M. citrifolia (5.94%) and S. suaveolens (5.78%). Stigmasterol was present at 1.25%, marginally lower than in Arabica coffee (1.74%) and M. citrifolia (2.74%). The quantity of palmitic acid methyl ester in H. orixense was more (1.15%) than that in Arabica coffee (0.48%). Heneicosane was identified at 1.02%, in contrast to 7.90% in M. citrifolia, indicating that this hydrocarbon is less prevalent in H. orixense. γ-Tocopherol levels were lower in H. orixense (0.59%) as compared to Arabica coffee (1.01%). These variations can be assigned to species-specific metabolic pathways, environmental factors, the maturity stage of leaves upon harvest, extraction techniques etc. Squalene, a triterpene possessing antioxidant, antimicrobial, antiinflammatory, antidiabetic and anticancer activities, is a significant component in several members of the Rubiaceae family, with concentrations ranging from modest (~14% in H. orixense and S. suaveolens) to exceptionally high (~41% in M. citrifolia). Quinic acid, a prominent phenolic acid derivative known for its antioxidant and antidiabetic properties, is prevalent in H. orixense, indicating its potential contribution to the plant's bioactivity profile. Likewise, the high concentration of 9-octadecenamide in H. orixense may indicate distinctive pharmacological potential, particularly in antibacterial and anti-inflammatory applications. These comparative data suggest that H. orixense possesses a fundamental set of bioactive compounds which is common with other Rubiaceae species, such as phytol, squalene, tocopherols, terpenes, and various fatty acid derivatives; however, the relative abundances vary significantly, influencing potential biological activities and chemotaxonomic characteristics. Overall, the wide range of pharmacological activities attributed to the identified compounds—including antioxidant, anti-diabetic, antimicrobial, anticancer, anti-inflammatory, hypocholesterolemic, etc. provide strong scientific backing to the ethnomedicinal use of H. orixense leaves in traditional medicine. The richness in terpenes, fatty acid esters, phenolic compounds, and phytosterols suggests a multifaceted therapeutic potential and supports further pharmacological investigations and drug development from this plant. Subsequent research should concentrate on isolating these phytocompounds and validating their activity via targeted in vitro and in vivo investigations. 5. Conclusion The present study provides comprehensive insight into the phytochemical composition of the methanolic extract of H. orixense leaves through preliminary screening and GC-MS analysis for the first time. The qualitative phytochemical screening confirmed the presence of key secondary metabolites, including phenolics, flavonoids, terpenoids, saponins, and alkaloids, indicating the reason behind plant's therapeutic potential. The GC-MS analysis revealed the presence of a wide spectrum of bioactive compounds, notably, terpenes, fatty acid derivatives, phenolic derivatives, and phytosterols. Many of these identified constituents such as phytol, squalene, α-tocopherol, β-sitosterol, and various methyl esters of fatty acids are known for their strong antioxidant, anti-inflammatory, antimicrobial, and anti-diabetic properties, as reported in previous scientific literature. The present findings are indirectly supporting the scientific validation of the traditional medicinal applications of H. orixense and establishing a phytochemical basis for its diverse biological activities. The chemical diversity observed in the methanolic leaf extract could be further investigated for pharmacological potential and natural product-based drug development. Future studies, including isolation, structural characterization, and in vivo evaluations are therefore, recommended to fully understand the therapeutic implications of the chemical compounds identified in the present study. Compliance with ethical standards Acknowledgments Authors express gratitude to the Advanced Instrumentation Research Facilities (AIRF) of Jawahar Lal Nehru University, New Delhi for providing facility of GC-MS for this investigation. Disclosure of conflict of interest Authors declare no conflict of interest in publication of this manuscript. GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 175 References [1] Chaachouay N, Zidane L. Plant-Derived Natural Products: A Source for Drug Discovery and Development. Drugs Drug Candidates. 2024; 3(1): 184-207. https://doi.org/10.3390/ddc3010011 [2] Ahmed SN, Ahmad M, Zafar M, Yaseen G, Iqbal N, Rashid N, Kousar S, Haroon A. Herbal Drugs: Safety, CostEffectiveness, Regulation, Current Trends, and Future Directions. In: Arunachalam, K., Yang, X., Puthanpura Sasidharan, S. (eds.) Bioprospecting of Tropical Medicinal Plants. Springer, Cham; 2023. https://doi.org/10.1007/978-3-031-28780-0_62 [3] Hunter W. Pharmacognostic Analysis of Medicinal Plants: Integrating Traditional Knowledge with Modern Science. J. Pharmacogn. Nat Prod. 2024; 10(3): 309-310. [4] Koche D, Shirsat R, Kawale M. An overview of major classes of phytochemicals: their types and role in disease prevention. Hislopia J. 2016; 9(1/2): 1-11. [5] Poongothai A. Qualitative and quantitative phytochemical analysis of Lantana camara leaf extract. J Emerg Technol Innov Res. 2019; 6(12): 603-607. DOI:10.13040/IJPSR.0975-8232.12(5).2755-64 [6] Kavitha R. Phytochemical screening and GC-MS analysis of bioactive compounds present in ethanolic extracts of leaf and fruit of Trichosanthesis dioica roxb. Int J Pharm Sci Res. 2021; 12(5): 2755-2764. DOI:10.13040/IJPSR.0975-8232.12(5).2755-64 [7] Dike CS, Emejulu AA, Chukwudoruo CS, Akpaki MA, Nsofor WN, Edom CV. GC-MS and FTIR analyses of bioactive compounds present in ethanol leaf extract of Sida acuta from Imo State, Nigeri. GSC Biol Pharmaceut Sci. 2023; 25(2):394-404. https://doi.org/10.30574/gscbps.2023.25.2.0500 [8] Prema V, Ramya R, Rukmanidevi D. Application of GC-MS in Phytochemical screening of Traditional Medicinal Plants - A Review. Int J Pharm Pharm Res. 2022; 25(3): 270-285. https://ijppr.humanjournals.com/wpcontent/uploads/2022/11/19.Prema-V-Ramya.-R-Rukmanidevi.-D.pdf [9] Nagaraja SK, Nayaka S, Kumar RS. Phytochemical Analysis, GC-MS Profiling, and In Vitro Evaluation of biological applications of different solvent extracts of Leonotis nepetifolia (L.) R.Br. Flower Buds. Appl Biochem Biotechnol. 2023; 195(2): 1197–1215. https://doi.org/10.1007/s12010-022-04201-2 [10] Anoor PK, Yadav AN, Rajkumar K, Kande R, Tripura C, Naik KS, Burgula S. Methanol extraction revealed anticancer compounds Quinic Acid, 2(5H) Furanone and Phytol in Andrographis paniculata. Mol Clin Oncol. 2022; 17(5): 151. https://doi.org/10.3892/mco.2022.2584 [11] Chakraborty P, Sasi S, Nair AA, Anjum N, Tripathi YC. Medicinal applications, phytochemistry and pharmacology of Hymenodictyon excelsum (Roxb) Wall: a review. Org Med Chem Int J. 2017; 2(3): 60-64.DOI: 10.19080/OMCIJ.2017.02.555589 [12] Jain SK. Dictionary of Indian Folk Medicine and Ethnobotany. New Delhi: Deep Publications; 1991. [13] Jain V, Jain SK. Compendium of Indian Folk Medicine and Ethnobotany (1991-2015). New Delhi: Deep Publications; 2016. [14] Suchaichit N, Kanokmedhakul S, Kanokmedhakul K, Moosophon P, Boonyarat C, Plekratoke K, Tearavarich R, Suchaichit NP. Phytochemical investigation and acetylcholinesterase inhibitory activity of bark of Hymenodictyon orixense. Nat Prod Res. 2018; 32(24): 2936–2939. https://doi.org/10.1080/14786419.2017.1389930 [15] Trease GE, Evans WC. Pharmacognosy. 15th ed. London, UK: Saunders Publishers; 2002. 42–44. [16] Tambe VD Bhambar RS. Estimation of Total Phenol, Tannin, Alkaloid and Flavonoid in Hibiscus tiliaceus Linn. Wood Extracts. Res rev: J Pharmacogn Phytochem. 2014; 2(4): 41-47. https://www.rroij.com/openaccess/estimation-of-total-phenol-tannin-alkaloid-and-flavonoid-in-hibiscus-tiliaceus-linn-wood-extracts- .php?aid=34291 [17] Chauhan A, Goyal MK, Chauhan P. GC-MS technique and its analytical applications in science and technology. J Anal Bioanal Tech. 2014; 5(6):1000222. DOI:10.4172/2155-9872.1000222 [18] Hwang IG, Kim HY, Woo KS, Lee SH, Lee J, Jeong HS. Isolation and Identification of the Antioxidant DDMP from Heated Pear (Pyrus pyrifolia Nakai). Prev Nutr Food Sci. 2013; 18(1): 76–79. https://doi.org/10.3746/pnf.2013.18.1.076 GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 176 [19] Chen Z, Xi G, Fu Y, Wang O, Cai L, Zhao Z, Liu Q, Bai B, Ma Y. Synthesis of 2,3-dihydro-3,5-dihydroxy-6-methyl-4Hpyran-4-one from maltol and its taste identification. Food Chem. 2021; 361: 130052. https://doi.org/10.1016/j.foodchem.2021.130052 [20] Mujeeb F, Bajpai P, Pathak N. Phytochemical evaluation, antimicrobial activity, and determination of bioactive components from leaves of Aegle marmelos. BioMed Res Int. 2014; 2014: 497606. https://doi.org/10.1155/2014/497606 [21] Kambouche N, Merah B, Bellahouel S, Bouayed J, Dicko A, Derdour A, Younos C, Soulimani R. Chemical composition and antioxidant potential of Ruta montana L. essential oil from Algeria. J Med Food. 2008; 11(3): 593-595. Doi:10.1089/jmf.2007.0515 [22] Gibka J, Kunicka-Styczyñska A, Gliñski M. Antimicrobial Activity of Undecan-2-one, Undecan-2-ol and Their Derivatives. J Essent Oil Bear Plants. 2009; 12(5): 605–614. https://doi.org/10.1080/0972060X.2009.10643763 [23] Lee S, Jena R, Odom AL. Substituted Pyridines from Isoxazoles: Scope and Mechanism. Org Biomol Chem. 2022; 33: 1-7. DOI: 10.1039/d2ob00779g [24] Das M, Prakash S, Nayak C, Thangavel N, Singh SK, Manisankar P, Devi KP. Dihydroactinidiolide, a natural product against Aβ25-35 induced toxicity in Neuro2a cells: Synthesis, in silico and in vitro studies. Bioorg Chem. 2018; 81: 340–349. https://doi.org/10.1016/j.bioorg.2018.08.037 [25] Das S, Koner A, Mobarak SH, Barik A. Attraction of the biocontrol agent, Lema praeusta, towards two Commelinaceae weed volatiles. J Appl Entomol Nov. 2021; 145(9): 869–889. doi: 10.1111/jen.12899. [26] Benali T, Bakrim S, Ghchime R, Benkhaira N, El Omari N, Balahbib, A, Taha D, Zengin G, Hasan MM, Bibi S, Bouyahya A. Pharmacological insights into the multifaceted biological properties of quinic acid. Biotechnol Genet Eng Rev. 2022; 40(4): 3408–3437. https://doi.org/10.1080/02648725.2022.2122303 [27] Fagbemi KO, Aina DA, Adeoye-Isijola MO, Naidoo KK, Coopoosamy RM, Olajuyigbe OO. Bioactive compounds, antibacterial and antioxidant activities of methanol extract of Tamarindus indica Linn. Sci Rep. 2022; 12(1): 9432. doi:10.1038/s41598-022-13716-x [28] Jayawardena TU, Kim H, Sanjeewa KKA, Kim S, Rho J, Jee Y, Ahn G, Jeon Y. Sargassum horneri and isolated 6hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (HTT); LPS-induced inflammation attenuation via suppressing NF-κB, MAPK and oxidative stress through Nrf2/HO-1 pathways in RAW 264.7 macrophages. Algal Res. 2019; 40: 101513. https://doi.org/10.1016/j.algal.2019.101513 [29] Ravitchandirane H, Singh V, Rajavel A, Sella RN. Characterization and metabolomic analysis of Plant-derived Extracellular Vesicles (PdEVs) isolated from indigenous medicinal plants. J App Biol Biotech. 2024; 12(6): 251260. DOI: 10.7324/JABB.2024.153542 [30] Jayashree I, Geetha D, Rajeswari M. GC-MS Analysis of bioactive constituents of Glochidion ellipticum WT. Int J Pharm Sci Res. 2015; 6(6): 2546-2550. DOI: 10.13040/IJPSR.0975-8232.6(6).2546-50 [31] Bano I, Deora GS. Preliminary phytochemical screening and GC-MS analysis of methanolic leaf extract of Abutilon pannosum (Forst. F.) Schlect. from Indian Thar desert. J Pharmacogn Phytochem. 2019; 8(1): 894-899. https://www.phytojournal.com/archives/2019.v8.i1.6866/preliminary-phytochemical-screening-and-gc-msanalysis-of-methanolic-leaf-extract-of-ltemgtabutilon-pannosum-ltemgtforst-f-schlect-from-indian-thar-desert [32] Avoseh ON, Mtunzi FM, Ogunwande IA, Ascrizzi R, Guido F. Albizia lebbeck and Albizia zygia volatile oils exhibit anti-nociceptive and anti-inflammatory properties in pain models. J ethnopharmacol. 2021; 268: 113676. https://doi.org/10.1016/j.jep.2020.113676 [33] Rajeswari G, Murugan M, Mohan VR. GC-MS analysis of bioactive components of Hugonia mystax L. (Linaceae). Res J Pharm Biol Chem Sci. 2012; 3(4): 301-308. https://www.rjpbcs.com/pdf/2012_3(4)/[32].pdf [34] Rahman MM, Ahmad SH, Mohamed MT, Ab Rahman MZ. Antimicrobial compounds from leaf extracts of Jatropha curcas, Psidium guajava and Andrographis paniculata. Sci World J. 2014; 2012: 635240. https://doi.org/10.1155/2014/635240 [35] Venkataravana LN, Uppin J, Ramanjineyulu NC, Krishna PG, Narayana JL. Discovering bioactive phytoconstituents from Citrullus lanatus for antimicrobial and antioxidants therapeutic applications. Eur J Med Chem Rep. 2024; 12: 100229. https://doi.org/10.1016/j.ejmcr.2024.100229. [36] Suresh A, Praveenkumar R, Thangaraj R, Oscar FL, Baldev E, Dhanasekaran D, Thajuddin N. Microalgal fatty acid methyl ester a new source of bioactive compounds with antimicrobial activity. Asian Pac J Trop Dis. 2014; 4(2): S979-S984. https://doi.org/10.1016/S2222-1808(14)60769-6 GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 177 [37] Abdel-Hady H, Abdel-Gawad MM, El-Wakil E. A. Characterization and evaluation of antioxidant activity of Ocimum canum leaves and its efficiency on Schistosoma mansoni larval stage. Indo Am. J Phar Res. 2017; 7(11): 978-994. https://iajpr.com/iajprfiles/uploaddir/171017.pdf [38] Mehta M, Puri R, Devi G, Angmo D, Boora P, Rani S. In Vivo Antidiabetic Activity and GC-MS Analysis of Ethanolic Extracts of Rabdosia rugosa (Wal. ex Benth.) H. Hara. Def Life Sci J. 2023; 8(4): 303-313. DOI: 10.14429/dlsj.8.18893 [39] Al-Marzoqi A, Hadi M, Hameed I. Determination of metabolites products by Cassia angustifolia and evaluate antimicrobial activity. J Pharmacogn Phytother. 2016; 8(2): 25-48. DOI:10.5897/JPP2015.0367 [40] Gopu C, Chirumamilla P, Daravath SB, Vankudoth S, Taduri S. GC-MS analysis of bioactive compounds in the plant parts of methanolic extracts of Momordica cymbalaria Fenzl. J Med Plants Stud. 2021; 9(3): 209-218. DOI: 10.22271/plants.2021.v9.i3c.1289 [41] Ukwubile CA, Ahmed A, Katsayal UA, Ya J, Mejida S. GC–MS analysis of bioactive compounds from Melastomastrum capitatum (Vahl) Fern. leaf methanol extract: An anticancer plant. Sci Afr. 2019; 3: e00059. https://doi.org/10.1016/j.sciaf.2019.e00059 [42] Mohamad OA, Li L, Ma JB, Hatab S, Xu L, Guo JW, Rasulov BA, Liu YH, Hedlund BP, Li W. J. Evaluation of the antimicrobial activity of endophytic bacterial populations from Chinese traditional medicinal plant licorice and characterization of the bioactive secondary metabolites produced by Bacillus atrophaeus against Verticillium dahliae. Front Microbiol. 2018; 9: 924. doi: 10.3389/fmicb.2018.00924 [43] Rautela I, Dheer P, Thapliyal P, Joshi T, Sharma N, Sharma M. GC-MS analysis of plant leaf extract of Datura stramonium in different solvent system. Eur J Biomed Pharm Sci. 2020; 5(10): 236-245. https://www.ejbps.com/ejbps/abstract_id/5071 [44] Idan SA, Al-Marzoqi AH, Hameed I. H. Spectral analysis and anti-bacterial activity of methanolic fruit extract of Citrullus colocynthis using gas chromatography-mass spectrometry. Afr J Biotechnol. 2015; 14: 3131-3158. DOI:10.5897/AJB2015.14957 [45] Huang ZR, Lin YK Fang JY. Biological and Pharmacological Activities of Squalene and Related Compounds: Potential Uses in Cosmetic Dermatology. Molecules. 2009; 14(1): 540-554. https://doi.org/10.3390/molecules14010540 [46] Amarowicz R. Squalene: A natural antioxidant? Eur J Lipid Sci Technol. 2009; 111: 411-412. DOI:10.1002/ejlt.200900102. [47] Ezhilan BP, Neelamegam R. GC-MS analysis of phytocomponents in the ethanol extract of Polygonum chinense L Pharmacogn Res. 2012; 4(1): 11–14. https://doi.org/10.4103/0974-8490.91028 [48] Sudha T, Chidambarampillai S, Mohan VR. GC-MS Analysis of Bioactive Components of Aerial parts of Fluggea leucopyrus Willd. (Euphorbiaceae). J Appl Pharm Sci. 2013; 3(5): 126-130. DOI: 10.7324/JAPS.2013.3524 [49] Khan IH, Javaid A. Anticancer, antimicrobial and antioxidant compounds of quinoa inflorescence. Adv Life Sci. 2020; 8(1): 68-72. https://www.als-journal.com/articles/vol8issue1/8111.20/943.pdf [50] Es-Sai B, Wahnou H, Benayad S, Rabbaa S, Laaziouez Y, El Kebbaj R, Limami Y, Duval RE. Gamma-Tocopherol: A Comprehensive Review of Its Antioxidant, Anti-Inflammatory, and Anticancer Properties. Molecules. 2025; 30(3): 653. https://doi.org/10.3390/molecules30030653 [51] Spikes AE, Paschen MA, Millar JG, Moreira JA, Hamel PB, Schiff NM, Ginzel MD. First contact pheromone identified for a longhorned beetle (Coleoptera: Cerambycidae) in the subfamily Prioninae. J Chem Ecol. 2010; 36(9): 943954. doi:10.1007/s10886-010-9837-8 [52] Rizvi S, Raza ST, Ahmed F, Ahmad A, Abbas, S, Mahdi F. The role of vitamin E in human health and some diseases. Sultan Qaboos Univ Med J. 2014; 14(2): e157–e165. https://pubmed.ncbi.nlm.nih.gov/24790736/ [53] Liao S, Omage SO, Börmel L, Kluge S, Schubert M, Wallert M, Lorkowski S. Vitamin E and Metabolic Health: Relevance of Interactions with Other Micronutrients. Antioxidants. 2022; 11(9): 1785. https://doi.org/10.3390/antiox11091785 [54] Choi JM, Lee EO, Lee HJ, Kim KH, Ahn KS, Shim BS, Kim NI, Song MC, Baek NI, Kim SH. Identification of campesterol from Chrysanthemum coronarium L. and its antiangiogenic activities. Phytother Res. 2007; 21(10): 954-959. doi:10.1002/ptr.2189 GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 178 [55] Choudhary D, Shekhawat JK, Kataria V. GC-MS Analysis of Bioactive Phytochemicals in Methanol Extract of Aerial Part and Callus of Dipterygium glaucum Decne. Pharmacogn J. 2019; 11(5): 1055-1063. DOI:10.5530/pj.2019.11.165 [56] Ashraf R, Bhatti HN. Chapter 10 - Stigmasterol, Ed(s): Mushtaq M, Anwar F. A Centum of Valuable Plant Bioactives, Academic Press; 2021. 213-232. ISBN 9780128229231. https://doi.org/10.1016/B978-0-12822923-1.00019-4. [57] Bakrim S, Benkhaira N, Bourais I, Benali T, Lee LH, El Omari N, Sheikh RA, Goh K W, Ming LC, Bouyahya A. Health Benefits and Pharmacological Properties of Stigmasterol. Antioxidants. 2022; 11(10): 1912. https://doi.org/10.3390/antiox11101912 [58] Gupta R, Sharma AK, Dobhal MP, Sharma MC, Gupta, RS. Antidiabetic and antioxidant potential of β-sitosterol in streptozotocin-induced experimental hyperglycemia. J Diabetes. 2011; 3(1): 29–37. https://doi.org/10.1111/j.1753-0407.2010.00107.x [59] Alappat L, Valerio M, Awad AB. Effect of vitamin D and β-sitosterol on immune function of macrophages. Int Immunopharmacol. 2010; 10(11): 1390–1396. https://doi.org/10.1016/j.intimp.2010.08.003 [60] Alvarez-Sala A, Attanzio A, Tesoriere L, Garcia-Llatas G, Barberá R, Cilla A. Apoptotic effect of a phytosterolingredient and its main phytosterol (β-sitosterol) in human cancer cell lines. Int J Food Sci Nutr. 2019; 70(3): 323–334. https://doi.org/10.1080/09637486.2018.1511689 [61] Nandi S, Nag A, Khatua S, Sen S, Chakraborty N, Naskar A, Acharya K, Calina D, Sharifi-Rad J. Anticancer activity and other biomedical properties of β-sitosterol: Bridging phytochemistry and current pharmacological evidence for future translational approaches. Phytother Res. 2024; 38(2): 592-619. https://doi.org/10.1002/ptr.8061 [62] Benito J, Marques G, Barro F, Gutiérrez A, Del Río JC, Rencoret J. Comprehensive Study of Lipophilic Compounds from Various Cereal Straws (Wheat, Triticale, Rye, and Tritordeum) ─ a Promising Source of Valuable Phytochemicals. J Agric Food Chem. 2025; 73(12): 7282–7297. https://doi.org/10.1021/acs.jafc.4c12445 [63] van Lierop B, Castle L, Feigenbaum A, Boenke A. Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionate. In: Spectra for the Identification of Additives in Food Packaging. Springer, Dordrecht; 1998. https://doi.org/10.1007/978-94-011-5222-8_55 [64] Neal-Kluever AP, Bailey AB, Hatwell KR. Safety assessment for octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)- propionate (CAS Reg. No. 2082-79-3) from use in food contact applications. Food Chem Toxicol. 2015; 86: 176190. https://doi.org/10.1016/j.fct.2015.10.004. [65] El-Sayed SE, Abdelaziz NA, El-Housseiny GS, Aboshanab KM. Octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate, an antifungal metabolite of Alcaligenes faecalis strain MT332429 optimized through response surface methodology. Appl Microbiol Biotechnol. 2020; 104(24): 10755–10768. https://doi.org/10.1007/s00253-020-10962-9 [66] Gupta S, Yadav M. K, Thangamani D, Vidhya C. S, Kalaimani P, Samuelraj J and Rajamony V. Herbal medicines: bridging traditional knowledge with modern pharmacology. Biochem. Cell. Arch., 2023; 23(1): 1577-1582. 10.51470/bca.2023.23.S1.1577. [67] Dar RA, Shahnawaz M, Ahanger MA, Majid I. Exploring the Diverse Bioactive Compounds from Medicinal Plants: A Review. J Phytopharmacol. 2023; 12(3): 189-195. DOI:10.31254/phyto.2023.12307 [68] Fais A, Era B. Phytochemical Composition and Biological Activity. Plants. 2024; 13(3): 331. https://doi.org/10.3390/plants13030331 [69] Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med. 2012; 12: 221. https://doi.org/10.1186/1472-688212-221 [70] Panche AN, Diwan AD, Chandra SR. Flavonoids: an overview. J Nutr Sci. 2016; 5: e47. https://doi.org/10.1017/jns.2016.41 [71] Vishe V, Kor N, Tarmale P, Sonawane P. Biological Activity of Flavonoids. Int J Pharm Sci. 2025; 3(6): 1672-1678. https://doi.org/10.5281/zenodo.15619295 [72] Rahman MM, Rahaman MS, Islam MR, Rahman F, Mithi FM, Alqahtani T, Almikhlafi MA, Alghamdi SQ, Alruwaili AS, Hossain MS, Ahmed M, Das R, Emran TB, Uddin MS. Role of Phenolic Compounds in Human Disease: Current Knowledge and Future Prospects. Molecules. 2021; 27(1): 233. https://doi.org/10.3390/molecules27010233 GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 156-179 179 [73] Sun W, Shahrajabian MH. Therapeutic Potential of Phenolic Compounds in Medicinal Plants-Natural Health Products for Human Health. Molecule. 2023; 28(4): 1845. https://doi.org/10.3390/molecules28041845 [74] Ramdani D, Yuniarti E, Jayanegara A, Chaudhry AS. Roles of Essential Oils, Polyphenols, and Saponins of Medicinal Plants as Natural Additives and Anthelmintics in Ruminant Diets: A Systematic Review. Animals. 2023; 13(4): 767. https://doi.org/10.3390/ani13040767 [75] Cosme F, Aires A, Pinto T, Oliveira I, Vilela A, Gonçalves BA. Comprehensive Review of Bioactive Tannins in Foods and Beverages: Functional Properties, Health Benefits, and Sensory Qualities. Molecules. 2025; 30(4): 800. https://doi.org/10.3390/molecules30040800 [76] Wei G, Kong L, Zhang J, Ma C, Wu X, Li X, Jiang H. Essential oil composition and antibacterial activity of Lindera nacusua (D. Don) Merr. Nat Prod Res. 2016; 30(23): 2704–2706. https://doi.org/10.1080/14786419.2015.1135145 [77] Jiangseubchatveera N, Liawruangrath B, Liawruangrath S, Teerawutgulrag A, Santiarworn D, Korth J, Pyne SG. The Chemical Constituents and the Cytotoxicity, Antioxidant and Antibacterial Activities of the Essential Oil of Graptophyllum pictum (L.) Griff. J Essent Oil Bear Plants. 2015; 18(1): 11–17. https://doi.org/10.1080/0972060X.2014.935036 [78] Li X, Xin Y, Mo Y, Marozik P, He T, Guo H. The Bioavailability and Biological Activities of Phytosterols as Modulators of Cholesterol Metabolism. Molecules. 2022; 27(2): 523. https://doi.org/10.3390/molecules27020523 [79] del Hierrro JN, Herrera T, Fornari T, Reglero G, Martin D. The gastrointestinal behavior of saponins and its significance for their bioavailability and bioactivities. J Funct Foods. 2018; 40: 484-497. https://doi.org/10.1016/j.jff.2017.11.032. [80] Sharma K, Kaur R, Kumar S, Saini RK, Sharma S, Powde SV, Kumar V. Saponins: a concise review on food related aspects, applications and health implications. Food Chem Adv. 2023; 2: 100191. https://doi.org/10.1016/j.focha.2023.100191. [81] Kytidou K, Artola M, Overkleeft HS, Aerts JMFG. Plant Glycosides and Glycosidases: A Treasure-Trove for Therapeutics. Front Plant Sci. 2020;11. DOI=10.3389/fpls.2020.00357 [82] Aryal B, Raut BK, Bhattarai S, Bhandari S, Tandan P, Gyawali K, Sharma K, Ranabhat D, Thapa R, Aryal D, Ojha A, Devkota HP, Parajuli N. Potential Therapeutic Applications of Plant-Derived Alkaloids against Inflammatory and Neurodegenerative Diseases. Evid Based Complement Alternat Med. 2022; 2022: 7299778. https://doi.org/10.1155/2022/7299778 [83] Ng YP, Or TC, Ip NY. Plant alkaloids as drug leads for Alzheimer's disease. Neurochem Int. 2015; 89: 260-270. https://doi.org/10.1016/j.neuint.2015.07.018 [84] Petri GL, Raimondi MV, Spanò V, Holl R, Barraja P, Montalbano A. Pyrrolidine in Drug Discovery: A Versatile Scaffold for Novel Biologically Active Compounds. Top Curr Chem. 2021; 379: 34. https://doi.org/10.1007/s41061-021-00347-5 [85] Onanuga AO, Okpala EO. Chemical Compositions and Antioxidant Activity of Volatile Oils from Morinda citrifolia and Beta vulgaris Leaves from Nigeria. Biol Med Nat Prod Chem. 2022; 11(2): 161-167. DOI: https://doi.org/10.14421/biomedich.2022.112.161-167 [86] Haruna A. GC-MS Profiling and Antifungal Activities of Morinda Citrifolia L. Leaf Extract Against Fungal Pathogens of Crown Rot Disease of Banana. J Phytol. 2023; 15: 132-38. https://doi.org/10.25081/jp.2023.v15.8423. [87] Danial ND, Asib N, Sadi T, Ismail SI. Evaluation of Morinda citrifolia Leaf Extract Against Phytophthora palmivora in Controlling Stem Canker on Durian (Durio zibethinus). Malays Appl Biol. 2025; 54(1): 24-37. https://doi.org/10.55230/mabjournal.v54i1.2947 [88] Jeon YA, Natraj P, Kim SC, Moon JK, Lee YJ. Comparative Analysis of Phytochemical and Functional Profiles of Arabica Coffee Leaves and Green Beans Across Different Cultivars. Foods. 2024; 13(23): 3744. https://doi.org/10.3390/foods13233744 [89] Lobo PD, Saraf A. Antioxidant Activity Study and GC-MS Profiling of Leaves, Stem and Root Extracts of Spermadictyon suaveolens Roxb. Acad J Biol. 2024; 46(3): 37–61. DOI: 10.15625/2615-9023/20624.