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GC-FID Phytochemical Profiling of Ethanolic Leaf Extract of Mentha piperita and Correlation of Identified Bioactive Constituents with Neuroprotective Activity

Chukwudi Francis Afuberoh¹,* Okoye Ogochukwu Fidelis², Anyaogu Charles Chinemeze², Ojimba Makuochukwu Immaculata³, Nonso Ikechukwu Odikpo², Ifechukwu Eucharia Obododike³, Onoriode Akpoghene Eyeghre¹, Chikwendu Sixtus Amadi¹

¹Department of Human Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, PMB 5001, Anambra, Nigeria.

²Department of Human Physiology, Faculty of Basic Medical Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli Campus, Anambra, Nigeria.

³Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Agulu Campus, PMB 5001, Anambra, Nigeria.

1. INTRODUCTION

Mentha piperita L. (peppermint), a member of the Lamiaceae family, is among the most widely cultivated and commercially significant aromatic plants globally. Its antioxidant, anti-inflammatory, antimicrobial, antispasmodic, and neuroprotective activities are well documented and largely attributed to its rich secondary metabolite profile (Iscan et al., 2002; Koşar et al., 2005). The essential oil and non-volatile fractions of M. piperita contain over 300 identified constituents, including terpenoids, flavonoids, phenolic acids, triterpenoids, and sterols. Their relative abundance varies with chemotype, geographic origin, extraction method, and plant maturity.

GC-FID is widely used for quantitative profiling of plant extracts because it is sensitive, reproducible, and compatible with volatile and semi-volatile compounds. It identifies compounds by retention time and quantifies them by peak area against external standards (Paton and Putievsky, 1996).

Alzheimer’s disease (AD) is a growing global health burden with few disease-modifying treatments. Aluminium chloride (AlCl₃) and D-galactose are established experimental inducers of AD-like neurotoxicity in rodents, producing oxidative stress, neuroinflammation, amyloid-beta (Aβ1-42) accumulation, tau hyperphosphorylation, and cognitive impairment. Identifying which phytoconstituents in M. piperita extract might plausibly drive its reported neuroprotective effects is therefore of both scientific and translational interest. This study presents the complete GC-FID phytochemical profile of ELMP and discusses how the identified constituents relate mechanistically to neuroprotective pathways relevant to AD, and to the peripheral hepatic and renal antioxidant outcomes reported in a companion in vivo study (Afuberoh et al., 2026).

2. MATERIALS AND METHODS

2.1 Plant Material and Extract Preparation

Fresh leaves of M. piperita were collected, authenticated by a botanist, and shade-dried at room temperature. The dried leaves were pulverised into a coarse powder using a mechanical blender. Ethanolic extraction was performed by soaking 500 g of powdered leaf material in 2.5 L of 95% ethanol for 72 hours with intermittent agitation. The mixture was filtered through Whatman No. 1 filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator at 40°C. The resultant crude extract was stored at 4°C in a sealed amber vial until analysis.

2.2 GC-FID Analysis

Phytochemical profiling was carried out by GC-FID (Agilent CHEM32, Instrument 1) on 18 February 2026 at the Department of Pharmaceutical Chemistry Laboratory, using an injection volume of 0.2 µL. The acquisition method (PHYTO-2023.M) used an external standard calibration protocol (Multiplier: 1.0000; Dilution: 1.0000). Peaks were identified by matching retention times with certified external reference standards. All compounds were assigned to Group 1 (TPH: Total Phenolic and Hydrocarbon fraction). Compounds below the limit of quantification were recorded as not detected (ND).

2.3 Rationale and Limitations of GC-FID for Flavonoid Detection

GC-FID was chosen because it allowed rapid, single-run detection across a broad polarity range using an existing in-house calibration library, from volatile monoterpenes to phenolic and flavonoid standards. However, GC-FID depends on analytes being sufficiently volatile, or chemically derivatised, to elute cleanly and respond reliably at the flame ionisation detector. Non-volatile, polyhydroxylated, or polymethoxylated compounds such as Myricetin and Nobiletin are prone to peak tailing, thermal degradation, and inconsistent response factors under GC conditions compared with liquid-phase methods. Accordingly, the flavonoid and phenolic acid quantities reported here should be read as an indicative screening-level fingerprint rather than a fully validated quantitative assay. Confirmatory analysis by LC-MS or HPLC-DAD, which does not require analyte volatility, is recommended before these values are used to support precise structure-activity conclusions.

3. RESULTS

3.1 GC-FID Phytochemical Profile

GC-FID analysis of ELMP identified 33 compounds across a retention time range of 3.925-31.656 minutes. Of these, 25 were quantified against external standards, totalling 79.52 ppm (Table 1). Nine compounds were detected as trace peaks below the quantification limit (ND). The chromatogram showed early-eluting terpenoid peaks dominating, with secondary flavonoid and phenolic acid peaks at higher retention times.

Table 1: Phytochemical Analysis of Ethanolic Leaf Extract of Mentha piperita Using GC-FID

RT: Retention Time; ND: Not Detected (below quantification limit). *Limonene detected at two retention time peaks (4.030 and 9.515 min); combined area and amount reported. Total quantified: 79.52 ppm.

Limonene, a monoterpene, eluted at two distinct retention times (4.030 and 9.515 min) with a combined peak area of 495.11 pA·s and a total quantified amount of 54.571 ppm (68.62% of all quantified phytochemicals). This dual elution may reflect limonene occurring in structurally distinct matrix environments, or co-eluting isomeric forms. Citral (5.266 ppm; 6.62%), an acyclic monoterpenoid aldehyde, eluted at 12.738 min. Myricetin (5.251 ppm; 6.60%), a polyhydroxylated flavonol, eluted at 14.607 min. Flavone (4.703 ppm; 5.91%) and Nobiletin (4.684 ppm; 5.89%) were the third and fourth most abundant flavonoid constituents. Epicatechin (3.883 ppm; 4.88%), Resveratrol (3.831 ppm; 4.82%), and Pinene (3.129 ppm; 3.94%) made up the remaining major constituents. Minor quantified constituents (≤1.17 ppm each) included Catechin, Daidzein, Vanillic acid, Flavon-3-ol, Naringin, Naringenin, Daidzin, Coumaric acid, Lunamarin, Kaempferol, Butein, Ferulic acid, Luteolin, Cinnamic acid, Baicalin, and Epigallocatechin. Nine compounds, including Ellagic acid, Gallocatechin 3-gallate, Robinetin, Tangeretin, Rosmarinic acid, Sinapinic acid, Syringic acid, Gentisic acid, and Piperic acid, were detected only as trace peaks (ND).

4. DISCUSSION

4.1 Terpenoid Constituents and Neuroprotective Relevance

Limonene’s dominance (68.62%) in the ELMP profile is a notable finding with clear pharmacological relevance. Limonene is a cyclic monoterpene with well-documented antioxidant, anti-inflammatory, anxiolytic, and neuroprotective effects in the literature. It scavenges free radicals directly, induces phase II detoxification enzymes, and activates the Nrf2/HO-1 antioxidant pathway. These known mechanisms offer a plausible chemical basis for the type of antioxidant benefit (e.g., reduced brain MDA, improved SOD and catalase activity) that would be expected in an AlCl₃/D-galactose model of AD-like neurodegeneration, and align with hepatic and renal antioxidant restoration reported in a related in vivo study by our group (Afuberoh et al., 2026). Limonene also inhibits NF-κB activation, which suppresses pro-inflammatory cytokines such as IL-1β and IL-6.

Citral (6.62%), the second most abundant quantified constituent, is an acyclic monoterpenoid aldehyde present as a mixture of geranial and neral isomers. It scavenges superoxide and hydroxyl radicals directly and inhibits the COX-2 and 5-LOX inflammatory pathways. Together, limonene, citral, and pinene make up approximately 79% of all quantified compounds, indicating that terpenoids are likely the extract’s primary antioxidant and anti-inflammatory contributors.

4.2 Flavonoid Constituents and Alzheimer’s Disease Pathology

Myricetin (6.60%) is a polyhydroxylated flavonol with neuroprotective mechanisms well characterised in the AD literature. It inhibits glycogen synthase kinase-3β (GSK-3β), a key kinase in tau hyperphosphorylation, and has been shown elsewhere to reduce amyloid-beta (Aβ) aggregation through direct interaction with Aβ fibrils. These literature-established mechanisms are consistent with the kind of reduction in Aβ1-42 and phosphorylated tau that would support ELMP’s reported neuroprotective activity. Myricetin also chelates aluminium ions, which is particularly relevant to limiting aluminium-induced oxidative damage in AlCl₃-based models.

Flavone (5.91%) and Nobiletin (5.89%) are established neuroprotective flavone constituents. Nobiletin, a polymethoxylated flavone, enhances CREB phosphorylation and promotes BDNF expression in prior studies, and separately inhibits β-secretase (BACE1) activity, reducing amyloidogenic APP processing. Epicatechin (4.88%) is a flavan-3-ol that inhibits acetylcholinesterase (AChE), which would be expected to help preserve synaptic acetylcholine levels. Resveratrol (4.82%), a stilbene polyphenol, activates SIRT1 deacetylase and AMPK signalling, mechanisms linked in the literature to autophagy-mediated clearance of aggregated Aβ and phosphorylated tau. Collectively, this flavonoid and polyphenol fraction offers a multi-target mechanistic rationale for the anti-amyloid, anti-tau, and cholinergic-protective potential of ELMP.

4.3 Phenolic Acids and Supporting Antioxidant Activity

Minor phenolic acid constituents identified in ELMP included Coumaric acid (0.409 ppm), Vanillic acid (0.550 ppm), Ferulic acid (0.204 ppm), Cinnamic acid (0.166 ppm), and trace levels of Rosmarinic, Syringic, Sinapinic, and Gentisic acid. Despite their low individual quantities, phenolic acids likely contribute to overall antioxidant capacity through synergistic radical scavenging and metal chelation. Ferulic acid is notable for its documented ability to cross the blood-brain barrier and inhibit both Aβ aggregation and AChE activity, with additional anti-inflammatory effects via NF-κB inhibition.

4.4 Dopaminergic and Neurotrophic Mechanisms

Kaempferol (0.312 ppm), Luteolin (0.187 ppm), Baicalin (0.158 ppm), Naringenin (0.425 ppm), and Naringin (0.474 ppm) are minor quantified constituents with mechanisms relevant to dopaminergic and neurotrophic support. Kaempferol protects dopaminergic neurons by scavenging reactive oxygen species and inhibiting monoamine oxidase B (MAO-B). Luteolin has been shown in prior work to enhance BDNF expression and TrkB receptor phosphorylation. Baicalin modulates the BDNF/TrkB/PI3K/Akt signalling cascade and suppresses neuroinflammatory pathways. Together with the dominant terpenoid and major flavonoid fractions, these minor constituents plausibly add to ELMP’s overall multi-target neuroprotective profile.

4.5 Trace Constituents and the Influence of Extraction Solvent

Nine compounds, including Ellagic acid, Gallocatechin 3-gallate, Robinetin, Tangeretin, Rosmarinic acid, Sinapinic acid, Syringic acid, Gentisic acid, and Piperic acid, were detected only as trace peaks (ND). This is notable for Rosmarinic acid, reported elsewhere as a major constituent of M. piperita dried leaf, typically 1.5-6.5% of dry weight (Petersen & Simmonds, 2003). Its near-absence here is unlikely to reflect a true absence of the compound in the source plant material, and more plausibly reflects the extraction conditions used. Rosmarinic acid is more polar than the dominant monoterpenes recovered in this extract, and 95% ethanol may under-extract it relative to more polar solvent systems (e.g., aqueous ethanol or methanol-water mixtures) commonly used in studies reporting high rosmarinic acid yields. Prolonged maceration and heat exposure during rotary evaporation may also promote degradation of rosmarinic acid, which is thermally and oxidatively labile. GC-FID’s limited sensitivity for non-volatile phenolic acids (Section 2.3) likely compounds this effect. Future work should compare solvent systems directly and pair GC-FID screening with LC-MS/HPLC confirmation to determine whether the low rosmarinic acid signal reflects extraction chemistry, analytical limitation, or genuine variation in the plant material.

5. CONCLUSION

The marked dominance of a single monoterpene (limonene), alongside a diverse but individually minor flavonoid and phenolic acid background, suggests that ELMP’s reported neuroprotective potential is unlikely to stem from one compound acting alone. The profile is instead consistent with a multi-constituent action model, in which a high-abundance terpenoid provides broad antioxidant and anti-inflammatory tone, while lower-abundance flavonoids (myricetin, nobiletin, epicatechin) may contribute more target-specific actions relevant to amyloidogenic and cholinergic pathology. This has a practical implication for future work: isolating and testing limonene alone would likely underestimate ELMP’s full neuroprotective potential, so fractionation studies should track flavonoid-enriched fractions separately from the terpenoid-rich volatile fraction.

The GC-FID limitations discussed in Section 2.3 mean this profile should be treated as a screening-level chemical fingerprint rather than a fully validated quantitative assay, particularly for the flavonoid and phenolic acid constituents. With that caveat, and pending LC-MS/HPLC confirmation and completion of correlative in vivo work, Mentha piperita remains a promising multi-constituent candidate warranting further mechanistic and eventual clinical evaluation in Alzheimer’s disease management.

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