All chemicals used were of analytical grade. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric chloride (FeCl₂, 4H₂O), acetylcholinesterase (AChE) type VI-S, from electric eel 349 U/mg solid, 5,5-dithiobis[2-nitrobenzoic acid] (DTNB), acetylthiocholine iodide (AChI), aluminum chloride, Iron sulphate, ferrozine, gallic acid, catechin, quercetin, (C₈-C₂₂) n-alkanes and tris[hydroxymethyl]aminomethane (tris buffer), potassium ferricyanide, trchloroacetic acid, and Folin-Ciocalteu reagent were supplied from Sigma Aldrich (Steinheim, Germany). Acetone, butanol, methanol and ethanol were obtained from Merck (supplied by VWR, France), while 2,5-dihydroxy benzoic acid and red phosphorous were purchased from Laser Bio Labs (France) and Sodium chloride (NaCl) was obtained from Carlo Erba Reactifs (SDS) (France).

In this study, the bracts and fruits of T. platyphyllos (Fig. 1) were investigated. The plant material was collected in El Feîdja National Park, located northwest of the town of Ghardimaou (Jendouba Governorate, Tunisia), at 36°29′10′′N and 8°18′55′′E. The samples were air-dried in the dark at room temperature for 7 days. Botanical identification was made by Pr. Ayda KHADHRI, Professor Associate at the Faculty of Sciences of Tunisia. A voucher specimen was deposited in the herbarium of Plant, Soil, Environment Interactions Laboratory, Department of Biology, Faculty of Sciences of Tunisia, under the registration number MALVACEAE-01.

The essential oil (EO) was obtained by hydrodistillation of 100 g of air-dried T. platyphyllos bracts and fruits using a Clevenger apparatus as per European Pharmacopeia guidelines. Following distillation, the oil was immediately extracted and stored in amber glass bottles at 4°C.

GC–MS analyses were conducted using an Agilent 6890 series gas chromatograph coupled with an Agilent 5973 N mass selective detector (Agilent Technologies, Little Falls, DE, USA). HP-5MS columns were employed for analysis, with helium as the carrier gas. The EO was diluted with n-hexane and injected into the GC-MS system. Identification of oil components was based on retention indices of n-alkanes (C₈-C₂₂), mass spectra comparison, and available literature [18].

In the current study, bracts and fruits of T. platyphyllos were dried at room temperature for 7 days before being ground separately in an ultra-centrifugal into powder using 0.2 mm particle size sieve. Ethanol extracts were prepared by shaking 100 g of each sample of T. platyphyllos in approximately 100 mL of ethanol for 72 h. The extracts were extracted at 4°C under reduced pressure using a rotary evaporator after being filtered through Whatmann No. 4 filter paper.

The antioxidant potential of ethanol extract and essential oils from T. platyphyllos was assessed using three tests: the DPPH radical scavenging test, the iron chelating capacity, and the iron reducing capacity.

The enzymatic activity of acetylcholinesterase in T. platyphyllos was assessed using a modification of Ellman’s method [26]. 30 mL of sample, 98 µL of 50 mM tris-HCl buffer pH = 8, and 7.5 µL of the acetylcholinesterase solution containing 0.26 U.mL⁻¹ were combined in an ELISA plate well and incubated for 15 min at 25°C. Then, 142 µL of 3 mM DTNB and 22.5 µL of an AChI solution were added. The absorbance was measured at 405 nm. A water control instead of the extract this having been assumed to be 100% active was carried out. The calculation of the percentage inhibition was carried out as follows and used for a control response.

I(%)=100−((Asample/Acontrol)×100),

where I is the percent inhibition of acetylcholinesterase, A_sample is the absorbance of the extract containing reaction and A_control is the absorbance of the reaction control. Tests were carried out in triplicate and a blank with Tris–HCl buffer instead of enzyme solution was used. Extract concentration providing 50% inhibition (IC₅₀) was obtained by plotting the inhibition percentage against extract concentrations.

Ethanol extracts of T. platyphyllos bracts and fruits were dissolved in acetone for analysis (4 mg.mL⁻¹, 50/50 volume), and the solutions were mixed with the matrix solution (10 mg.mL⁻¹ in acetone). The matrix, which facilitates the deposition of the sample in the instrument, was 2,5-dihydroxy benzoic acid. Calibration was done with red phosphorous. Ion formation was enhanced by adding to the matrix a concentrated solution of sodium chloride (NaCl) (10 mg.mL⁻¹ in distilled water). The mix used for examination consisted of 3 parts each of the samples and matrix solutions, added of 1 part NaCl solution. 0.5–1 µL of the mix so obtained and soluble in 98% acetone was then placed on the MALDI target. The residue remaining on the filter paper was further dissolved with water. Thus, a tannin so prepared was extracted in successive operations using ethyl acetate and 98% acetone.

MS Excel and SPSS softwares were used for the relevant statistical analysis. Statistical test was investigated using IBM SPSS 20, and an interesting degree of significance (p < 0.05) was obtained. All the analyses were carried out in triplicates.

A total of 23 compounds were identified by chromatographic studies using GC-MS analysis of the essential oils of T. platyphyllos bracts and fruits (Table 1). According to their percentages and retention indices (RI), the components were determined with respect to (C₈-C₂₂) n-alkanes. They are presented in Table 1 in the order of their elution on the HP-5 column.

Different classes have been formed by dividing the identified compounds (Table 1). Oxygenated monoterpenes were the dominant class (77.79%–90.64%), with p-menth-1-en-4-ol making up the majority of the Tilia fruits (66.05%) and cis-linalool oxide forming up the majority of the bracts (19.51%). The hydrocarbon monoterpenes constituted the second class (8.14%–8.46%). Hydrocarbon sesquiterpenes, on the other hand, were minor substances with a mean proportion below 0.4%.

We measured the amounts of total polyphenols, flavonoids, and proanthocyanidins in ethanolic extracts of T. platyphyllos bracts and fruits.

A significant variance in the extracts’ phenolic levels was found, as shown in Table 2. In the fruit extract, polyphenols represented a high content (7.65 mg GAE.g⁻¹ DW), while flavonoids and proanthocyanidins constituted the least amount (3.01 mg Q.g⁻¹ DW and 1.11 mg CE.g⁻¹ DW, respectively). Additionally, the fruit extract’s total phenolic content was substantially higher than that of the bract extract (4.81 mg GAE.g⁻¹ DW). These findings indicate that the concentration of phenolic chemicals varied considerably with the organs.

Table 2 shows the total antioxidant capacity of T. platyphyllos fruits, bracts, ethanolic extract, and essential oils. The variation of the concentration (IC₅₀) that causes 50% inhibition is summarized in the results.

According to the organs (fruits and bracts), T. platyphyllos ethanolic extract and essential oils showed a significant variance in their capacity to scavenge free radicals. The ethanolic extract’s activity was found to be lowest in the bracts organs (1.22 mg.mL⁻¹), and the highest was found in the essential oils of the fruits and bracts (0.52 and 0.91 mg.mL⁻¹, respectively).

It is possible to draw the conclusion that antiradical activity is influenced by the type of extract and organs based on the data obtained. The results obtained showed that the antioxidant activity of fruit essential oils is higher than that of ethanol extract.

The iron-reducing power was determined based on the effective concentration (EC₅₀). The linear regression line between the concentration and the related optical density is used to calculate the EC₅₀ values. The extract’s low reduction power and high EC₅₀ value are related. Thus, it can be inferred from Table 2 that the essential oil of fruits has the highest reducing power (0.67 mg.mL⁻¹).

The ability level for iron chelation was employed to evaluate the chelation capacity of metal ions by the plant extracts. Fruit essential oil has the strongest chelating power and the lowest IC₅₀ (2.03 mg.mL⁻¹). In comparison to ethanolic extract, the essential oil had the maximum antioxidant capacity, according to the data. The high concentration of monoterpenes that have been hydrocarbonated and oxygenated may be the cause of the essential oil’s potent antioxidant properties.

By inhibiting the enzyme acetylcholinesterase (AChE), which is the key for degrading acetylcholine, a variety of neurological illnesses, such as senile dementia, Alzheimer’s, myasthenia gravis and ataxia, appear that can be treated [27,28]. According to Howes et al. [29], plants were previously utilized to improve cognitive performance and treat various symptoms of Alzheimer’s disease. Galantamine, one of the enzyme inhibitors extracted from snowdrop extract, is utilized to make the majority of medications used in Alzheimer’s therapy [27]. The literature-recommended approach was modified to determine the level of AChE inhibition [30].

The activity of ethanolic extracts of fruits and bracts from T. platyphyllos ability to inhibit acetylcholinesterase was examined (Table 2). The results showed IC₅₀ values varying between 0.77 and 1.39 mg.mL⁻¹. These results show that all extracts have modest inhibitory efficacy and are comparable to those obtained with other aromatic and medicinal herbs [31–34].

The essential oil from T. platyphyllos bracts had the most potent inhibitory effect (IC₅₀ = 0.77 mg.mL⁻¹), followed by the essential oil from fruits (IC₅₀ = 0.95 mg.mL⁻¹). Results analysis reveals that these oils have a mild inhibitory effect on AChE. According to Orhan et al. [28], the IC₅₀ value for the drug galantamine, which is utilized pharmacologically, is 1 mg.mL⁻¹. The monoterpenes molecule is responsible for this inhibitory effect because the extract has greater activity than the AChE inhibitor. The IC₅₀ values of ethanolic extracts from fruits and bracts are 1.23 and 1.39 mg.mL⁻¹, respectively.

From the known literature this appears to be the first time that inhibition of the AChE enzyme in vitro is reported by using essential oils and ethanolic extracts.

Based on the detection of MALDI-TOF spectra, it was shown that the bracts and fruit extracts of T. platyphyllos included a range of phenolic compounds (Table 3 and Figs. 2, 3). The chemical profiles of the ethanolic extracts of bracts and fruits are substantially similar, with a few small exceptions.