Abstract

The chemical profiles of methanolic extract from the leaves, trunk, and roots of Nauclea diderrichii were determined by liquid chromatography coupled with mass spectrometry (LC-MS). Total polyphenol content was determined using the Folin-Ciocalteu reagent method, and total flavonoid content using the aluminum trichloride method. Antioxidant potential was evaluated via DPPH free radical scavenging, ferric reducing antioxidant power (FRAP), and HPTLC bioautography. Extraction yields were highest for the roots (13.17%) and leaves (12.68%). Total polyphenol content was highest in the leaves (270.68 mg GAE/g), followed by the roots (244.26 ± 0.01 mg GAE/g). Total flavonoid content followed the same trend as total polyphenol content; specifically, the leaves (142.27 mg QE/g) contained higher levels than the roots (29.03 ± 0.01 mg QE/g). Quinic, chlorogenic, protocatechuic, and caffeic acids were identified in the leaves. In contrast, the trunk and roots were rich in triterpene compounds such as quinovic, quafrinoic, and emmolic acids. Antioxidant potential was most pronounced in the leaves (DPPH (IC₅₀) = 90.90 ± 0.03 µg/mL; FRAP = 814.25 ± 0.08 mg BHA-E/g) and roots (DPPH (IC₅₀) = 92.16 ± 0.04 µg/mL; FRAP = 723.36 ± 0.03 mg BHA-E/g).

Keywords

Nauclea diderrichii, LC-MS, HPTLC Bioautography, Polyphenol, Flavonoid, Antioxidant Potential

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1. Introduction

The genus Nauclea (Rubiaceae) comprises seven species distinguished by the shape of their placentas, stipules, and fruit type [1], as well as leaf shape, seed margins, petiole length, calyx and corolla pubescence, flowering head diameter, and leaf pubescence [2]. The best-known species are Nauclea latifolia Smith, Nauclea pobeguinii (Hua ex Pobég.) Merr. and Nauclea diderrichii (De Wild.) Merr. These species are distributed across the tropical and subtropical zones of Central Africa (Republic of the Congo, DRC, Gabon, Angola, and the Central African Republic) and West Africa (Benin, Senegal, and Côte d’Ivoire). Other species, such as Nauclea vanderguchtii (De Wild.) Petit, Nauclea gilletii (De Wild.) Merr., and Nauclea xanthoxylon (Chev.) Aubr., are less well known [3]. One species, Nauclea nyasica, is found in East Africa, in countries such as Tanzania, Mozambique, and Malawi. Among these species, N. latifolia has been the most extensively studied [4]-[7]. Nauclea species are used in traditional African medicine to treat various ailments, as evidenced by the development of three improved traditional antimalarial medicines based on N. latifolia [8], N. latifolia combined with Cassia occidentalis [9] [10], and N. pobeguinii [11]-[13].

Given the interest surrounding Nauclea species, we focused our attention on Nauclea diderrichii, which is known by several vernacular names such as “bilinga”, “aloma”, “badi”, and “kusia”. This species can reach heights of 30 to 40 m, with a trunk diameter ranging from 0.9 to 1.5 m. Its fruit is edible [14]. N. diderichii is used as a medicinal plant within its distribution range to treat various conditions [15]-[19]. In Nigeria, a decoction of the stem bark is used to treat anemia, stomach aches, and indigestion, while a leave decoction is used as a lotion for measles [20]. The leaves are used to treat gastrointestinal [21] and skin infections [22]. In Benin, Cameroon, and Côte d’Ivoire, the species is used to treat malaria [23] [24].

N. diderichii extracts are also used in traditional medicine to treat microbial and respiratory ailments [25]. The literature cites several phytochemical and pharmacological studies conducted on N. diderichii. Nwodo and Agbo [20] demonstrated the antitrypanosomal activity of the stem bark; this extract contained alkaloids, tannins, saponins, and flavonoids. Aderibigbe et al. [26] confirmed the anthelmintic activity of the leaves. N. diderichii specimens acclimatized in the Republic of the Congo have not yet been the subject of phytochemical or pharmacological studies. The aim of this study is to determine the phytochemical profiles and evaluate the antioxidant potential of Nauclea diderichii extracts.

2. Materials and Methods

2.1. Plant Material

The plant material was collected in the Patte d’Oie forest (04˚16'12.85'' South and 015˚14'58.92'' East) in Brazzaville in April 2025. The species was authenticated by botanists from the National Herbarium, and a specimen was preserved under reference ND-01. The various parts of the same plant, namely the leaves, trunk bark, and root bark were dried separately for two weeks, away from sunlight. The samples were then ground into a very fine powder.

2.2. Extraction

50 g of powder (consisting of leaves, trunk bark, or root bark) was macerated in 300 mL of methanol under magnetic stirring for 24 hours. The mixture was filtered, and the resulting filtrate was evaporated to dryness under vacuum using a Heidolph rotary evaporator.

2.3. Determination of the Chemical Profiles

The phytochemical profiles of the extracts were obtained using the method described by Boungou-Tsona et al. [27]. This method is based on liquid chromatography coupled with mass spectrometry. Analyses were performed on an Ultimate 3000 RSLC UHPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) coupled with a quaternary rapid separation pump (Ultimate autosampler) and a diode array detector (DAD). The UHPLC system was connected to an Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with electrospray ionization in positive (ESI+) or negative (ESI−) mode. Compounds were eluted on an Uptisphere Strategy C18 column (250 × 4.6 mm, 5 µm; Interchim, Montluçon, France). The mobile phase consisted of a mixture of water and 0.1% (v/v) formic acid (phase A) and a mixture of acetonitrile and 0.1% (v/v) formic acid (phase B). The phase A gradient was programmed as follows: 100% (0 min), 80% (10 min), 73% (35 min), 0% (40 - 50 min), and 100% (51 - 60 min). The flow rate was 0.8 mL/min, and the injection volume was 5 µL. The spray voltage was set to 3 kV, the capillary temperature to 320˚C, and the auxiliary gas temperature to 400˚C, with flow rates (in arbitrary units) of 2, 50 (for nitrogen), and 10 (for sheath gas). Collision energy ranged from 10 to 50 eV. Full-scan spectra were acquired at a resolution of 70,000, and MS² spectra at a resolution of 17.500. Xcalibur software version 4.6 (Thermo Fisher Scientific Inc., MA, USA) was used for spectral processing.

2.4. Total Polyphenol Content (TPC)

Total polyphenol content was determined using the method described by Sokmen et al. [28], with minor modifications. 0.1 g sample of each material (leaves, trunk bark, and root bark) was dissolved in 50 mL of distilled water. A 300 µL aliquot of this solution was mixed with 1.5 mL of Folin-Ciocalteu reagent (diluted 1:10 v/v); four to five minutes later, 1.2 mL of a 7.5% (w/v) sodium carbonate solution was added. After 2 hours of incubation in the dark, absorbance was measured at 760 nm against a blank using a Jenway spectrophotometer. Experiments were performed in triplicate. A gallic acid calibration curve was used to determine total polyphenol content, expressed as milligrams of gallic acid equivalent per gram of dry matter (mg GAE/g).

2.5. Total Flavonoid Content (TFC)

Total flavonoid content was determined according to the method described by Arvouet-Grand et al., with minor modifications [29]. 0.1 g sample of plant extract was dissolved in 100 mL of distilled water. Four milliliters of distilled water were added to a 1 mL aliquot of this solution. Several solutions were then added successively to the resulting mixture: at t = 0 minutes, 0.3 mL of sodium nitrite solution (NaNO₂; 5%); and after 5 minutes, 0.3 mL of aluminum chloride solution (AlCl₃; 10%). After 6 minutes, 2 mL of sodium hydroxide solution (NaOH; 1 N) and, finally, 2.4 mL of distilled water were added. The mixture was then incubated for 30 minutes. Absorbance was measured at 510 nm against a blank using a Jenway UV-visible spectrophotometer. The experiments were performed in triplicate. Total flavonoid content was determined using a quercetin calibration curve and is expressed as mg quercetin equivalents per gram of dry matter (mg QE/g).

2.6. Antioxidant Activity Assay

- DPPH free radical scavenging assay

The quantitative evaluation of anti-radical activity was performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method described by Brand-Williams et al. [30] and Meda et al. [31], with minor modifications. 300 µL of methanolic extract solutions at various concentrations (25, 50, 75, and 100 µg/mL) were mixed with 3 mL of a methanolic DPPH solution (0.18 mM, Sigma-Aldrich). Reaction kinetics were monitored for 45 minutes at 517 nm using a Jenway UV-visible spectrophotometer. Measurements were taken against a methanol blank. Ascorbic acid, used as a reference, was treated in the same manner as the extracts. Inhibition percentages (I%) were calculated using the following equation:

$I\%=\frac{AbsC-AbsE}{AbsC}\times 100$

The 50% inhibitory concentration was calculated based on the percentage of inhibition using the formula:

$I{C}_{50}=\frac{Concentration\times 50}{I\%}$

The experiments were repeated three times, and the results are presented as the mean ± SEM.

- Ferric Reducing antioxidant Power assay (FRAP)

The iron-reducing antioxidant power was determined using the method described by several authors, with minor modifications [32]. This method is based on the reduction of ferric iron (Fe³⁺) in the potassium hexacyanoferrate complex [K₃Fe(CN)₆] to ferrous iron (Fe²⁺), resulting in a color change of the solution from yellow to blue. To 0.4 mL of each sample (at 200 µg/mL), 1 mL of phosphate buffer (0.2 M; pH 6.6) and 1 mL of potassium hexacyanoferrate ([K₃Fe(CN)₆], 1%) were added. After a 30-minute incubation at 50˚C in a water bath, 1 mL of trichloroacetic acid (10%) was added. The resulting mixture was centrifuged at 3000 rpm for 10 minutes; the supernatant was collected and then mixed with 0.2 mL of ferric chloride (FeCl₃, 0.1%). The resulting mixture was left to stand in the dark for 30 minutes. A blank was prepared in the same manner but without the extract. Absorbance was measured at 700 nm using a Jenway UV-visible spectrophotometer. A calibration curve for butylated hydroxyanisole (BHA) was used to determine the antioxidant power of the extracts. Experiments were performed in triplicate. Results are expressed as mg BHA equivalent/g dry matter (mg EBHA/g).

2.7. Analysis of Extracts by HPTLC-Bioautography

Analyses were performed on 20 x 10 cm glass plates coated with 0.20 mm silica gel 60 (Merck, Darmstadt, Germany). The equipment used included a CAMAG HPTLC system (Muttenz, Switzerland) equipped with an automatic TLC sampler (ATS4), an automatic developing chamber (ADC2) with humidity control, a TLC visualizer, a CID III plate immersion device (for derivatization), and a heating plate. Vision CATS software was used for data processing. Extract solutions were prepared in HPLC-grade methanol at a concentration of 5 mg/mL. The elution system consisted of: dichloromethane/ethyl acetate/formic acid/acetic acid/water (25:100:10:10:11 v/v/v/v/v). After elution, the plates were immersed in solutions of the derivatizing agents NP (1 g in 150 mL of ethyl acetate) and PEG 400 (5 g in 150 mL of ethyl acetate), then dried at 120˚C and visualized under UV light at 254 nm and 366 nm. A separate plate was prepared using the same procedure but was derivatized with a DPPH· solution (50 mg of DPPH· in 150 mL of methanol).

3. Results and Discussion

3.1. Yield

The extracts were obtained by maceration of the plant material in methanol. The extraction yields obtained were 12.68 ± 0.07, 4.46 ± 0.02, and 13.17 ± 0.06 for the leaves, trunk, and roots, respectively (Table 1). The roots yielded the highest return compared to the other plant parts. Other authors reported a yield of 21.0% for the trunk, which is significantly higher than our results [33]. Similarly, Isa et al. [34] obtained a much lower yield for the leaves (3.47%) compared to our study.

3.2. Total Polyphenol and Total Flavonoid Contents

Phenolic compounds, particularly flavonoids, are considered an important class of phytochemicals. They are widely distributed in plants. Flavonoids are known for their numerous pharmacological properties [35]. Indeed, they can neutralize free radicals, thereby exhibiting antioxidant activity [36]. Figure 1 presents the total polyphenol and total flavonoid contents of the various parts of Nauclea diderichii. The assay results are summarized in Table 1. Regarding total polyphenols, the content obtained were 270.68 ± 0.04 mg GAE/g, 131.95 ± 0.08 mg GAE/g, and 244.26 ± 0.01 mg GAE/g for the leaves, stem, and roots, respectively. The leaves were richest in total polyphenols, followed by the roots. Regarding flavonoids, the contents obtained were 142.27 ± 0.05 mg QE/g, 46.3 ± 0.04 mg QE/g, and 120.72 ± 0.01 mg QE/g for the leaves, stem, and roots, respectively. As with the total polyphenol assay, the leaves were rich in total flavonoids.

Table 1. Extraction yields from different parts of Nauclea diderichii.

^aExpressed as mg gallic acid equivalents/g of dry plant material (mg GAEs/g); ^bExpressed as mg quercetin equivalents/g of dry plant material (mg QEs/g); ^cExpressed as µg/mL; ^dExpressed as mg butylhydroxyanisole equivalents/g of dry plant material (mg BHAs/g).

3.3. Chemical Profiles

LC-MS analysis in negative ESI mode of the leaves, trunk, and roots of Nauclea diderrichii enabled the detection of several phytochemical compounds. All identified compounds are listed in Table 2. The compounds were identified based on experimentally obtained fragment ions by comparing them with fragment ions from laboratory and online databases. The chemical profile of the leaves (Figure 1(A)) shows the following compounds: chlorogenic acid, protocatechuic acid, glucose, mannitol, quinic acid, citric acid, and lophirone A. In the stem (Figure 1(B)), glucose, glucuronic acid, galactonic acid, quinic acid, caffeic acid, quinovic acid, emmolic acid, and quafrinoic acid were found. In the root extract, glucose, quinic acid, rhyncoside B, quinovic acid, and emmolic acid were identified (Figure 1(C)).

Table 2. Phytochemical composition of methanolic extracts from the leave, trunk, and roots of Nauclea diderichii.

+: presence of the compound in the extract; −: absence of the compound in the extract.

Figure 1. Chemical profiles of Nauclea diderichii extracts, Leaves (A), trunk (B), Root (C).

Analysis of the results shows that the leaves extract is rich in constituents commonly found in plant extracts, except for lophirone A, a biflavonoid isolated from the species Lophira lanceolata [37]. Regarding the stem and root extracts, triterpenoid compounds predominate (See Table 2). These compounds have previously been identified in extracts of N. diderrichii and other Nauclea species [18] [38]. Indeed, quinovic acid and quafrinoic acid have been identified in the trunk bark of N. diderrichii acclimatized in Gabon and Nigeria [16] [17] [39]. Quinovic acid has also been identified in the root bark of N. pobeguinii acclimatized in the Democratic Republic of the Congo [40]. Emmolic acid has been identified in extracts of Ziziphus jujuba var. spinosa [41] and Emmenospermum alphitonioides [42]. Quafrinoic acid has been isolated from N. pobeguinii [43], N. xanthoxylon [44], and Quassia africana [45]. Several alkaloids have been isolated and identified in extracts of N. diderrichii [46] [47]. The phytochemical diversity observed in Nauclea species, particularly N. diderrichii, may explain their multiple pharmacological properties. The structures of the main compounds identified in the various extracts are shown in Figure 2.

Figure 2. Main compounds identified in the methanolic extracts of organs of Nauclea diderrichii.

3.4. Antioxidant Activity

The antioxidant activity of the extracts was evaluated using two methods: DPPH free radical scavenging and iron reduction. Results were compared to those of ascorbic acid for the DPPH method. For the FRAP method, results were expressed based on a butylhydroxyanisole calibration curve. Analysis of the results shows that the leaf extract (IC₅₀ = 90.90 ± 0.03 µg/mL) is slightly more active than the root extract (IC₅₀ = 92.16 ± 0.04 µg/mL), regardless of the method used (Table 1). These results align with the total polyphenol and total flavonoid contents; indeed, the leaves and root extracts with higher total polyphenol and total flavonoid levels exhibited the highest activity. Several studies have highlighted the antioxidant potential of Nauclea diderrichii extracts [52] [53]. The compounds present in the leaves, and root extracts are likely responsible for this antioxidant activity. Phenolic acids such as quinic, chlorogenic, protocatechuic, and caffeic acids, which predominate in the leaves extract are thought to contribute to the antioxidant potential of the various extracts [43] [54] [55]. The antioxidant potential observed with the root extract is likely attributable to the presence of triterpene acids, specifically quinovic acid [56] and emmolic acid [57].

3.5. HPTLC-Bioautography of the Extracts

The use of high-performance thin-layer chromatography (HPTLC) combined with NP/PEG and DPPH is an approach that enables the rapid determination of the phytochemical profile and the individual contribution of constituents to the biological potential of a plant matrix [58]. Figure 3 presents the results obtained from the analysis of leaves, trunk bark, and root extracts of N. diderrichii. Analysis of Figure 3(A) shows that all extracts visualized at 366 nm exhibit nearly identical phytochemical profiles, characterized by the presence of light blue, dark blue, and brown, fluorescent bands typical of phenolic compounds, particularly flavonoids. Additionally, bright red, fluorescent bands are observed in the leaf extract, likely indicating the presence of plant pigments. Analysis of Figure 3(B), following derivatization with DPPH, reveals stronger antioxidant potential in the leaf and root extracts, characterized by yellow bands against a purple background.

Figure 3. HPTLC analysis with derivatization using NP/PEG at 366 nm (A) and DPPH· under white light (B).

4. Conclusion

The aim of this study was to determine the chemical profiles and evaluate the antioxidant potential of methanolic extracts from various parts of Nauclea diderrichii, a species endemic to Republic of the Congo. The results indicate higher yields for the leaves and roots. Both plant parts are also rich in total polyphenols and total flavonoids. Analysis of the chemical profiles reveals that the leaves are rich in phenolic acids, whereas the trunk and roots are rich in triterpene compounds, specifically emmolic, quafrinoic, and quinovic acids. Extracts from the leaves and roots demonstrated significant antioxidant potential, characterized by low inhibitory concentrations. These findings could substantiate the numerous uses of Nauclea diderrichii in traditional African phytotherapy. Consequently, it would be worthwhile investigating other pharmacological activities of these extracts, particularly their anti-inflammatory, anticancer, antidiabetic, and antimicrobial properties.

Conflicts of Interest

The authors declare no conflicts of interest.

References