# In-depth analysis of Strychnos innocua Delile using UHPLC-MS/MS metabolomics tools consolidated with advanced separative techniques

Mawaté Florance Brukum, Isabelle Schmitz, Carlos Afonso, Abdelhakim

Elomri, Kafui Kpegba, Kodjo Selom Evenamede, Amegnona Agbonon,

Oudjaniyobi Simalou

► To cite this version:

Mawaté Florance Brukum, Isabelle Schmitz, Carlos Afonso, Abdelhakim Elomri, Kafui Kpegba, et al. In-depth analysis of Strychnos innocua Delile using UHPLC-MS/MS metabolomics tools consolidated with advanced separative techniques. Next Research, 2025, 4, pp.101217. 10.1016/j.nexres.2025.101217. hal-05508748

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# In-depth analysis of *Strychnos innocua* Delile using UHPLC-MS/MS metabolomics tools consolidated with advanced separative techniques

Mawaté Florance Brukum<sup>a,b</sup>, Isabelle Schmitz<sup>b,# ID</sup>, Carlos Afonso<sup>b ID</sup>, Abdelhakim Elomri<sup>b,* ID</sup>, Kafui Kpegba<sup>a</sup>, Kodjo Selom Evenamede<sup>a ID</sup>, Amegnona Agbonon<sup>c</sup>, Oudjaniyobi Simalou<sup>a ID</sup>

## ARTICLE INFO

### Keywords:

*Strychnos innocua* Delile

Metabolomics

Molecular networks

Iridoid

## ABSTRACT

*Strychnos innocua* Delile is a small tree in the Loganiaceae family. In Africa, it is used for a variety of pharmacological purposes. In particular, the roots are used in Togo to manage sickle cell disease. In recent years, developments in analytical methods and processing tools have made it possible to explore natural products of real industrial and pharmaceutical interest. In this study, the secondary metabolites of *S. innocua* root and stem barks were investigated using UHPLC-MS/MS and semi-preparative HPLC. Analytical techniques and bioinformatics tools were combined in a high-throughput methodology enabling plant extracts to be dereplicated. Methanolic and hydroethanolic extracts were analysed by UHPLC-MS/MS, using an ESI-Q/Orbitrap mass spectrometer. The raw data was pre-processed using MZmine2 and then the MS/MS data was exported to build molecular networks via the Global Natural Products Social platform. In parallel, semi-preparative HPLC was used to isolate the predominant secondary metabolites. We were able to annotate 150 and 118 compounds respectively in the root and stem bark extracts. Several secondary metabolites were annotated in the *S. innocua* root and stem bark extracts, including chlorogenic acid, O-caffeoylquinic acid, luteolin and yohimbine. Extracts of root bark were found to be rich in iridoids and those of stem bark in polyphenols. Semi-preparative HPLC enabled us to isolate and characterise by 1D and 2D NMR an iridoid, sweroside, and a monoterpene alkaloid, bakankoside, the main compounds in the root extracts. The results reveal a richness in secondary metabolites in *Strychnos innocua* Delile, which could explain its biological activities.

## 1. Introduction

For centuries and even millennia, our ancestors have used plants to relieve pain, heal ailments and dress wounds. Medicinal plants contain secondary metabolites that represent a major source of molecules that can be used by humans in the field of pharmacology. Medicinal plants have historically proven their value as reservoirs of compounds with therapeutic potential, and still represent an important pool for the identification of novel drug leads [1]. Despite advances in modern pharmacology, their therapeutic use remains widespread in many parts of the world, particularly in developing countries [2]. In these regions, medicinal plants offer a more accessible alternative to conventional pharmaceuticals, whose high costs and limited availability often restrict their use [3].

The Togolese flora contains a multitude of plants used by the population to treat various pathologies, such as *S. innocua* Delile (*S. innocua*) [4], which belongs to the Loganiaceae family. In Africa, it is used for a variety of pharmacological purposes [5]. Its roots are used in northern Nigeria to treat skin infections of candidiasis and other diseases [6]. In Togo, according to ethnobotanical surveys, the roots of this plant are traditionally used to treat sickle cell anaemia [7]. In order to provide scientific support for this traditional use, our research team previously investigated the anti-sickling activity of the root and stem bark extracts of *S. innocua* [8]. The study confirmed the plant's ability to significantly reduce erythrocyte falciformation, and revealed notable levels of polyphenols and flavonoids, as well as slight antioxidant activity. These results provided a scientific basis for the present study, which aimed to explore the chemical diversity of *S. innocua*. Despite its traditional uses,

<footnote><sup>a</sup> Laboratory of Organic Chemistry and Natural Substances, LabCOSNat, Faculty of Sciences, Lomé 1515, Togo</footnote>

<footnote><sup>b</sup> Normandie Univ, UNIROUEN, INSA Rouen, CNRS, CARMeN (UMR 6064) ex COBRA, Rouen 76000, France</footnote>

<footnote><sup>c</sup> Laboratory of Physiology/Pharmacology, Faculty of Sciences, University of Lome 01 BP 1515 Lome 01, Lome, Togo</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: hakim.elomri@univ-rouen.fr (A. Elomri).</footnote>

<footnote># Present address: Univ Rouen Normandie, INSA Rouen Normandie, CNRS, Normandie Univ, PBS UMR 6270, F-76000, Rouen, France.</footnote>
few chemical studies have been carried out on this plant. A study carried out on the bark of the root revealed the presence of alkaloids, terpenes, saponins, tannins, flavonoid and steroids [6]. To date, only a few common compounds from the plant kingdom have been described, such as umbelliferone, campesterol and β-sitosterol from the ethyl acetate extract of root bark using a conventional extraction, isolation and purification technique [9].

In recent years, developments in analytical methods and processing tools have made it possible to explore biological sources and natural products of real industrial and pharmaceutical interest. Metabolomics research has evolved, as has one of its techniques, high-performance liquid chromatography (HPLC), which is effective in separating compounds according to their polarity. Coupling this technique with high-resolution mass spectrometry enables a large number of compounds to be screened. In order to better explore the polar extracts of *Strychnos innocua*, a survey of secondary metabolites was carried out using Ultra-High Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-MS/MS). The aim of this work is to adapt the non-targeted metabolomics workflow to the characterisation of this species. Analytical techniques and bioinformatics tools were combined in a high-throughput methodology useful for dereplicating extracts from this plant.

## 2. Material and methods

### 2.1. Plant material

The plant was collected in April 2021 in Kanté in the Kara region of Togo. It was identified by the Botany Department of the Faculty of Science at the University of Lomé under number TOGO15914. The stem and root barks of the plant were harvested, washed and dried in the laboratory at room temperature. They are then ground in a mill (Thomas Muler series 4, reference 3375 E20).

### 2.2. Extracts preparation

The various extractions were carried out at a rate of 100 g of plant material for 1 L of solvent (ethanol/water and methanol) by maceration during 72 h.

Firstly, the powder (root and stem barks) was macerated directly in the ethanol/water mixture (80:20 v/v).

Secondly, a two-step protocol was applied with a solvent gradient. Firstly, the powder (root and stem barks) was macerated in dichloromethane to obtain the apolar extract for subsequent analysis. The residual powder was then macerated with methanol to extract the polar compounds. After evaporation to dryness, hydroethanolic extracts (21.86 g for root bark and 15.68 g for stem bark), methanolic extracts (16.67 g for root bark and 11 g for stem bark) and dichloromethane extracts (2.67 g for root bark and 1.43 g for stem bark) were obtained. Hydroethanolic and methanolic extracts were used for analysis by UHPLC-MS/MS. The methanolic extracts were also used to separate and purify certain compounds.

### 2.3. Analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

#### 2.3.1. Protocol for preparing samples for analysis

Samples for analysis were prepared using LC-MS purity solvents. The hydroethanolic and methanolic extracts of stem bark were solubilised in water to obtain a stock solution with a concentration of 10 mg/mL. The hydroethanolic and methanolic extracts of root bark were solubilised in methanol. These solutions were then diluted in water for the stem barks and in a methanol/water mixture (7:3, v/v) for the root barks. Leading to a concentration of 2 mg/mL and then 200 µg/mL. The samples were centrifuged, and the supernatant recovered for analysis. The samples were then analysed by UHPLC-MS/MS using solutions at a concentration

of 200 µg/mL.

#### 2.3.2. Analysis of samples

Liquid chromatography was carried out using a UHPLC chromatography system (Vanquish, Thermo Scientific). The samples are placed in an automatic sampler thermostated at 6 °C, which allows for the automatic injection of 2 µL of each sample following the defined sequences. The various metabolites in our extracts were then separated according to their polarity using a reverse-phase C18 column (ACQUITY UHPLC HSS T3 Column, 100 Å, 1.8 µm, 2.1 mm × 100 mm equipped with a 0.2 µm prefilter), while being thermostated at 45 °C. The LC-MS grade solvents used as mobile phases were water + 0.1 % formic acid (lane A) and acetonitrile + 0.1 % formic acid (lane B). The solvent flow rate was set to 0.4 mL/min with a stepwise linear gradient. The gradient started at 5 % B and remained at this level for 0.5 min. After 0.5 min, the gradient shifted to 65 % B at 8 min and then to 100 % B at 11 min; this condition was maintained until 14.5 min. The gradient returned to the initial situation (5 % B) at 15 min and then stabilised until the end of the experiment at 19 min.

The different separated metabolites were then detected using a hybrid quadrupole / Orbitrap mass spectrometer (Exploris™ 120, Thermo Scientific) equipped with an electrospray ionisation source. Samples are analysed in both positive and negative ionisation modes, for a range of m/z 100 to 1500. For each mass spectrum, the 3 most intense precursor ions are fragmented. The source parameters were: Transfer tube temperature: 320 °C, Desolvation temperature: 350 °C, Capillary voltage: 3.5 kV in positive mode; 3.0 kV in negative mode, Sheath gas: 25 Arb, Aux gas: 10 Arb; Sweep gas: 1 Arb; Orbitrap resolution in MS mode: 60,000, RF Lens: 70 %, EASY-IC mode: On; MS/MS mode: stepped HCD Collision energies: 15, 40, 60 %, Orbitrap resolution: 15 000.

Mass calibration is performed externally before analysis and internally in real time during analysis using the EASY IC mode. The internal calibrant is fluoranthene (PAH) with a negative ionisation mode ion at m/z 201.25 and a positive ionisation mode ion at m/z 203.27. Mass measure precision is most of the time better than 5 ppm.

#### 2.3.3. Processing of data obtained by UHPLC-MS/MS

The raw data obtained in UHPLC-MS/MS are imported into MZmine2 for processing. The aim of this tool is to extract the list of compounds present in each extract. The masses were detected using the Exact mass algorithm in the mass tab, which were then used to construct chromatograms using the ADAP chromatogram builder algorithm [10]. Chromatograms are created after this operation and form lists of features characterised by *m/z* ratios, retention times, peak area and associated fragmentation data. These chromatograms are then deconvoluted to eliminate background signals, separate isomers and link MS/MS data with the corresponding features [11]. Isotopes and compounds detected in duplicate get removed.

The lists of features obtained after this process are then analysed. The chemical formulas are assigned automatically via the Formula prediction module according to parameters following the seven golden rules [12]. Source adducts and fragments are annotated with the corresponding modules, which compare MS and MS/MS data. Detailed parameters and corresponding numerical values are given in Table S1.

After processing in MZmine2, the data is imported into the GNPS platform to create molecular networks using the Feature Based Molecular Networking (FBMN) algorithm [13]. Using FBMN, molecular families of metabolites are created based on the similarity of their MS/MS spectra. This algorithm also highlights isomers belonging to the same family and provides relative quantitative details. The MS/MS spectra were annotated using the in silico dereplication tool. Here is the paragraph provided automatically by the website after network construction, with the numerical parameters indicated: "A molecular network was created with the Feature-Based Molecular Networking (FBMN) workflow) on GNPS (https://gnps.ucsd.edu). MS/MS spectra were window filtered by choosing only the top 6 fragment ions in the ± 50 Da window throughout the
spectrum. The precursor ion mass tolerance was set to 0.02 Da and the MS/MS fragment ion tolerance to 0.02 Da. A molecular network was then created where edges were filtered to have a cosine score above 0.7 and >4 matched peaks. Further, edges between two nodes were kept in the network if and only if each of the nodes appeared in each others respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set to 100, and the lowest scoring edges were removed from molecular families until the molecular family size was below this threshold. The spectra in the network were then searched against GNPS spectral. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks."

Cytoscape was used to visualise and graphically design the molecular networks [14].

## 2.4. Isolation of major metabolites from root bark extract

The aim of this section is to isolate the main constituents of our extract in their pure state. These compounds, which are not commercially available, will be used as reference standards to consolidate our annotations.

30 g of methanolic root bark extract was separated directly by reversed-phase medium-pressure liquid chromatography using an Intechim colonne PF-15C18HP-F0040. Elution was carried out using mixtures of water (A) and methanol (B) with increasing polarity to give 13 large fractions (F1 to F13). Analytical HPLC analysis was then carried out on these fractions to visualise their chromatographic profiles, allowing fraction F3 (150 mg) to be chosen for further separation. Fraction F3 (named H1081) was separated by semi-preparative HPLC (Puri Flash® 4250, 200 bar) and was eluted using a mixture of water (A) and acetonitrile (B). The gradient started at 10 % B at *t* = 0 and increased linearly to 55 % B at *t* = 45 min and then to 100 % B at *t* = 55 min. This separation led to the isolation of two pure compounds named C1 (6 mg) and C2 (10 mg). The two isolated products were lyophilised and analysed by mass spectroscopy and 1D-2D NMR.

## 2.5. Statistics

The relative abundance of each class of secondary metabolites (e.g., flavonoids, iridoids, alkaloids, phenolic acids) was estimated using a statistical approach. Specifically, for each class, the number of compounds annotated was divided by the total number of annotated metabolites in the extract to calculate the proportion (%) of each chemical family. The calculations and graphical representations were performed using GraphPad Prism 8.

# 3. Results and discussion

The main objective of our study was to explore the chemical diversity of *Strychnos innocua* extracts, focusing on polar extracts from root and stem barks, as these had previously demonstrated significant biological activity, including anti-sickling effects, in our earlier work [8]. UHPLC-MS/MS analysis was used to establish a detailed metabolic map of the crude extracts using bioinformatics tools [13], providing an initial overview of their chemical composition. In this study, advanced separation techniques including low and medium-pressure semi-preparative HPLC were employed in combination with UHPLC-MS/MS high-throughput metabolomic analysis. Both were used to isolate and structurally characterise the major constituents. This integrated approach, which combines modern metabolomics with classical natural product chemistry, reflects the current frontier of plant chemistry research and enhances the reliability of compound annotation. This complementary approach led to the annotation of two compounds at the highest confidence level (level 1 according to the recommendations of the Metabolomics Standards Initiative [15]), laying the foundations for future pharmacological investigations.

## 3.1. Analysis by UHPLC-MS/MS

Hydroethanolic and methanolic extracts of root and stem barks were analysed by UHPLC-MS/MS. For the analysis, the data was processed using MZmine2 as summarises in Fig. 1. During this processing, background noise, blank elements, adducts, isotopes and complexes were eliminated. After this step, the hydroethanolic and methanolic extract data for each organ (root and stem bark) and the blank data were aligned. The aligned data were then exported to build the molecular networks using the GNPS online platform. The alignment and construction of the molecular networks made it possible to annotate the features corresponding to the compounds present in our various extracts. The annotations were provided in accordance with the Metabolomics Standard Initiative, in which level 1 corresponds to compound identification, level 2 to putative annotations, level 3 to putative chemical class annotations and level 4 to unknown molecules [15].

## 3.2. Secondary metabolites annotated in root bark extracts

Comparative analysis of hydroethanolic and methanolic extracts of root bark by UHPLC-MS/MS was carried out in positive and negative ionisation mode. This made it possible to obtain chromatograms of the data in positive mode and negative mode (Figure S1). Fig. 2 shows the molecular network with at least two bound compounds, obtained using GNPS and visualised in Cytoscape for positive mode data. Figure S2 and S3 show the complete molecular network in positive and negative mode respectively. The annotations were assigned by comparison with online databases (KEGG, HMDB) and completed by the study of the molecular network (GNPS). The analysis detected a total of 423 (positive mode) and 193 (negative mode) features in our extracts. These features were then annotated to identify around 150 metabolites (Table S2). This represents a substantial proportion, especially considering the inherent complexity and challenges associated with metabolite annotation in natural extracts. All the major compounds in the methanolic extract were also found in the hydroethanolic extract and are summarised in Table 1.

For example, the annotation of lattice A (Fig. 3) was achieved using all these tools. Pre-processing with the MZmine2 software of the raw data obtained in positive mode identified two features *m/z* 358.1498 and *m/z* 359.1339 as bakankoside (monoterpene alkaloid) and sweroside (secoiridoid) respectively, by consulting the databases. After processing the data on the GNPS platform, the two compounds are found in the same molecular network and are linked to other unidentified features. By comparing the MS/MS spectra of the identified and unidentified compounds and thanks to an additional PubChem search, we were able to annotate *m/z* 521.1868 swertiapunimarin, *m/z* 491.1761 apifuranosylsmeroside derivative and *m/z* 375.1288 swertiamarin. The other features in the network have odd molecular masses with MS/MS spectra similar to that of bakankoside, which enabled them to be annotated as alkaloids.

The chemical families of all the annotated metabolites were then deduced and used to produce Fig. 4A.

## 3.3. Secondary metabolites annotated in stem bark extracts

Analysis of the hydroethanolic and methanolic extracts of the stem bark gave a chromatogram in positive mode and negative mode (Figure S4) with the data pretreated in MZmine2. Figure S5 and S6 show the complete molecular network in positive and negative mode respectively. Features annotation was used to identify 118 compounds (Table S3). The identified metabolites were classified according to their decreasing peak area and the major ones were recorded in Table 2. The two extracts have almost all the major metabolites in common. The chemical family of all the annotated metabolites was then deduced and used to produce a diagram (Fig. 4B).
<figure><img src="image_2.png" /><figcaption>Fig. 1. Processing of data obtained by UHPLC-MS/MS.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2. Molecular networks with at least two bound compounds constructed by GNPS from the positive mode data of our extracts. The colour corresponds to the origin of the sample (red: hydroethanolic extract; blue: methanolic extract). The node labels display the *m/z* ratio. The size of the nodes is proportional to the intensity, and the size of the edges to the cosine score.</figcaption></figure>

## 3.4. Analysis of metabolites identified in strychnos innocua extracts

### 3.4.1. Analysis of metabolites identified in root barks

The compounds identified in the root bark extracts (Table 1) indicate the presence of the carbohydrate family (α-D-mannoheptulopyranose), iridoids (more specifically secoiridoids, e.g., sweroside, gardenoside, swertiamarin, barlerin), alkaloid (cinchonine, bakankoside), flavonoids (pinocembrin rhamnosylglucoside, dalpanin, Feruloyl-genipin gentio-bioside derivative), steroids (2α-(Hydroxymethyl)-5α-androstane-3β,17β-diol) and carboxylic acid (quinic acid, 9,12,13-Trihydroxyocta-dec-10-enoic acid) (Fig. 5). These results are in line with the phyto-chemical screening carried out on root powder [8].

According to Fig. 4A, based on the annotated metabolites, the various root bark extracts are richer in iridoid families. More specifically, iridoid glucosides and secoiridoids, followed by flavonoids and alkaloids.

### 3.4.2. Analysis of metabolites identified in stem bark extracts

The UHPLC-MS/MS analysis carried out on the stem bark extracts showed that the methanolic and hydroethanolic extracts had a similar chemical profile. According to the results in Table 2, the two extracts contain several common secondary metabolites, including phenolic acids (chlorogenic acid, O-caffeoylquinic acid, Feruloylquinic acid derivative), flavonoids (luteolin-O-glucoside, luteolin, pinocembrin 7-rhamnosylglucoside, quercetin, Phellamurin), carbohydrates, iridoids (swertiamarin, secologanine, monotropein), and alkaloids (yohimbine,

aspidofractin, acetylajmaline derivatives) (Fig. 5). Fig. 4B shows that the bark of the stem is rich in polyphenolic compounds, more specifically flavonoids and phenolic acids. These two sub-families, phenolic acids and flavonoids, are well known for multiple pharmacological activities, antifungal, antioxidant, antitumor and antihypertensive [16,17].

### 3.4.3. Iridoids

In our study, iridoid compounds represented 27.33 % of all annotated compounds in the root bark and 12.7 % in the stem bark, clearly indicating the greater richness of the root extracts in iridoids. Iridoids are monoterpenes characterised by a cyclopenta[c]pyran skeleton, sometimes referred to as iridane [18]. The main members of this family are iridoid glucosides, such as secoiridoids. The latter are biogenetically derived from iridoids by oxidative opening of the C7-C8 bond of the genin. Later research revealed the presence of iridoids in several species of the genus *Strychnos*, including *Strychnos nux-blanda*, *Strychnos nux-vomica*, *Strychnos spinosa* and others [19–21]. This pattern suggests that iridoid biosynthesis is a chemical characteristic shared by certain species in this genus. Thus, the presence of iridoids in our *Strychnos innocua* extracts seems entirely credible and compatible with existing phytochemical studies [5].

Numerous studies have focused on the biological and pharmacological activities of iridoids and secoiridoids. An in-depth study of the pharmacological activities of isolated iridoids and secoiridoids revealed that many of them are the active ingredients of plants used in traditional medicines [22]. It has also been shown that iridoid glycosides produced
<table><caption>**Table 1**
Majority metabolites identified from analysis of hydroethanolic (HE) and methanolic (MeOH) extracts of *S. innocua* root bark.</caption><thead><tr><th>No peak</th><th>Rt</th><th>Name of proposed metabolite</th><th>m/z Observed</th><th>MS2(+) MS2(-)</th><th>Error (ppm)</th><th>Type of ion</th><th>Molecular Formula</th><th>Chemical family</th><th>Extract</th><th>AL</th></tr></thead><tbody><tr><td>1</td><td>0.60</td><td>α-D-Mannoheptulopyranose</td><td>209.0667</td><td>119.0349, 101.0241, 89.0240, 71.0143, 59.0136</td><td>0.2</td><td>[M-H]<sup>-</sup></td><td>C<sub>7</sub>H<sub>14</sub>O<sub>7</sub></td><td>Carbohydrate</td><td>HE; MeOH</td><td>2</td></tr><tr><td>2</td><td>0.62</td><td>Quinic acid</td><td>191.0561</td><td>191.0558, 127.0406, 85.0295, 93.0347</td><td>0.1</td><td>[M-H]<sup>-</sup></td><td>C<sub>7</sub>H<sub>12</sub>O<sub>6</sub></td><td>Carboxylic acid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>3</td><td>3.05</td><td>Swertiamarin</td><td>373.1136 375.1288</td><td>356.1312, 213.0760, 195.0666, 177.0547, 107.0496</td><td>-1.1 0.5</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>16</sub>H<sub>22</sub>O<sub>10</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>4</td><td>3.06</td><td>Bakankoside</td><td>358.1498</td><td>358.1498, 196.0980, 126.0549</td><td>0.4</td><td>[M + H]<sup>+</sup></td><td>C<sub>16</sub>H<sub>23</sub>NO<sub>8</sub></td><td>Alkaloid</td><td>HE; MeOH</td><td>1</td></tr><tr><td>5</td><td>3.43</td><td>Sweroside</td><td>359.1339</td><td>197.0808, 179.0690, 127.0393, 111.0806, 97.0281</td><td>0.6</td><td>[M + H]<sup>+</sup></td><td>C<sub>16</sub>H<sub>22</sub>O<sub>9</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>1</td></tr><tr><td>6</td><td>3.43</td><td>Gardenoside</td><td>403.1242</td><td>403.1243, 225.9960, 195.0653, 179.0573, 125.0254</td><td>-0.9</td><td>[M-H]<sup>-</sup></td><td>C<sub>17</sub>H<sub>24</sub>O<sub>11</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>7</td><td>3.59</td><td>Coumaroyl genipin gentiobioside derivative</td><td>695.2193</td><td>695.2233, 463.1260, 339.1091, 163.0399, 145.0290, 119.0509</td><td>0.1</td><td>[M-H]<sup>-</sup></td><td>C<sub>32</sub>H<sub>40</sub>O<sub>17</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>3</td></tr><tr><td>8</td><td>3.67</td><td>Pinocembrin 7-rhamnosylglucoside</td><td>563.1767 565.1918</td><td>371.1314, 177.0548, 145.0286, 117.0326, 89.0388</td><td>-0.5 0.3</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>27</sub>H<sub>32</sub>O<sub>13</sub></td><td>Flavonoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>9</td><td>3.77</td><td>7-acetyloxy-5-hydroxy-7-methyl-1-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylic acid</td><td>433.1347</td><td>433.1304, 397.1269, 225.0766, 155.0356, 111.0452</td><td>-1</td><td>[M-H]<sup>-</sup></td><td>C<sub>18</sub>H<sub>26</sub>O<sub>12</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>10</td><td>3.92</td><td>Cinchonine</td><td>295.1806</td><td>295.1799, 277.1703, 144.0813, 138.0921, 120.0801,</td><td>0.3</td><td>[M + H]<sup>+</sup></td><td>C<sub>19</sub>H<sub>22</sub>N<sub>2</sub>O</td><td>Alkaloid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>11</td><td>4.29</td><td>Barlerin</td><td>447.1505</td><td>447.1553, 401.1405, 239.0909, 169.0505, 59.0137</td><td>-0.6</td><td>[M-H]<sup>-</sup></td><td>C<sub>19</sub>H<sub>28</sub>O<sub>12</sub></td><td>Iridoid</td><td>HE</td><td>2</td></tr><tr><td>12</td><td>4.45</td><td>Cyclopenta[c]pyran-4-carboxylic acid, 1,4a,5,7a-tetrahydro-7-(hydroxymethyl)-1-[6-O-[6-O-[22]-3-(4-hydroxyphenyl)-1-oxo-2-propen-1-yl]-beta-D-glucopyranosyl]-β-D-glucopyranosyl]oxy-, methyl ester</td><td>695.2193 697.2345</td><td>501.1799, 341.1202, 323.1122, 147.0438,</td><td>695.2230, 515.1616, 339.1090, 163.0400, 145.0290</td><td>0.1 0.9</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>32</sub>H<sub>40</sub>O<sub>17</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>13</td><td>4.46</td><td>O-Cinnamoylgentiobiosyl-genipin</td><td>681.2394</td><td>501.1664, 371.1316, 325.1299, 195.0665, 131.0487</td><td>0.7</td><td>[M + H]<sup>+</sup></td><td>C<sub>32</sub>H<sub>40</sub>O<sub>16</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>3</td></tr><tr><td>14</td><td>4.87</td><td>Feruloyl-genipin gentiobioside derivative</td><td>725.2299</td><td>679.2178, 373.1120, 179.0566, 147.0457, 59.0247</td><td>0.1</td><td>[M-H]<sup>-</sup></td><td>C<sub>33</sub>H<sub>42</sub>O<sub>18</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>3</td></tr><tr><td>15</td><td>5.47</td><td>Dalpanin</td><td>533.1662 535.1814</td><td>371.1401, 341.1202, 203.0859, 147.0437, 119.0485</td><td>-0.4 0.7</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>26</sub>H<sub>30</sub>O<sub>12</sub></td><td>Flavonoid</td><td>HE; MeOH</td><td>2</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>No peak</th><th>Rt</th><th>Name of proposed metabolite</th><th>m/z Observed</th><th>MS2(+) MS2(-)</th><th>Error (ppm)</th><th>Type of ion</th><th>Molecular Formula</th><th>Chemical family</th><th>Extract</th><th>AL</th></tr></thead><tbody><tr><td>16</td><td>6.42</td><td>9,12,13-Trihydroxyoctadec-10-enoic acid</td><td>329.2333</td><td>329.2308, 211.1347, 171.1015, 139.1134, 99.0812</td><td>-0.1</td><td>[M-H]</td><td>C<sub>18</sub>H<sub>34</sub>O<sub>5</sub></td><td>Carboxylic acid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>17</td><td>11.33</td><td>2αl-(Hydroxymethyl)-5α-androstane-3β,17β-diol</td><td>323.2582</td><td>277.2147, 179.1440, 135.1168, 93.0702, 71.0858,</td><td>0.3</td><td>[M + H]<sup>+</sup></td><td>C<sub>20</sub>H<sub>34</sub>O<sub>3</sub></td><td>Steroid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>18</td><td>11.85</td><td>3β-Hydroxy-4β-methyl-5α-cholest-7-ene-4α-carboxylate</td><td>445.3678</td><td>445.3698, 427.3677, 289.2186, 165.0916, 69.0700,</td><td>0.3</td><td>[M + H]<sup>+</sup></td><td>C<sub>29</sub>H<sub>48</sub>O<sub>3</sub></td><td>Sterol</td><td>HE</td><td>2</td></tr></tbody></table>

Rt: Retention time; AL: Annotation Level; HE: Hydroethanolic; MeOH: Methanolic.

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Cytoscape display of molecular network results based on GNPS data. Example of annotation of networked compounds. Methanolic extract (blue node) and hydroethanolic extract (red node).</figcaption></figure>

by plants mainly protect against infection by viruses and micro-organisms and rapidly repair damaged areas [23]. According to several studies, these compounds have anti-inflammatory, anti-allergic [24], hypotensive, anti-tumour, hypoglycaemic, antiviral, antibacterial, hepatoprotective and antioxidant activities: [25,26]. Gardenoside is a natural compound found in *Gardenia* fruit that has hepatoprotective properties. It suppresses the pain of chronic constriction lesions by regulating P2×3 and P2×7 receptors [27,28]. Similarly, barlerin, natural compound found in *Barleria prionitis* [29], showed moderate glutathione S-transferase (GST) inhibitory activity with an IC₅₀ value of 38.9 mM. It also exhibited low acetylcholinesterase (AChE) inhibitory activity with an IC₅₀ of 100.0 mM, and low free radical scavenging capacity, with an IC₅₀ greater than 5 mg/ml [29]. Swertiamarin is a glycosidic secoiridoid with several pharmacological activities. It is anti-inflammatory and antioxidant, and has other properties [30,31]. The analgesic potential of swertiamarin was reported by Jaishree et al. [32]. They examined the in vivo antinociceptive activity of swertiamarin in mice and found that treatments with this molecule significantly increased the latency period on a hot plate [32].

### 3.4.4. Flavonoids

The annotated metabolites indicate the same proportion of flavonoids (18 %) in our stem bark and root bark extracts. Flavonoids are a large group of polyphenolic compounds with a benzo-γ-pyrone structure

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Circular diagram of chemical families of metabolites annotated in hydroethanolic and methanolic extracts of root bark (4A) and stem bark (4B) based on UHPLC-MS/MS analysis.</figcaption></figure>

and are ubiquitous in plants. They are synthesised via the phenylpropanoid pathway [33]. They are known for their multiple biological activities, mainly linked to their polyphenolic structure. Flavonoids are known to contribute to overall health thanks to their antioxidant properties, which translate into effective action on obesity, such as reducing body weight, body fat and plasma triglycerides/cholesterol. These beneficial effects are linked to reduced fat absorption, increased energy expenditure and modulation of lipid metabolism [34]. Quercetin has been reported to possess several biological activities. Studies have shown that quercetin prevents various diseases, such as osteoporosis, cancer, tumours, lung disease and cardiovascular disease. The antioxidant effects of quercetin play an important role in the prevention and treatment of all these diseases [35]. According to meta-analyses of randomised trials, quercetin appears to improve total cholesterol, LDL-cholesterol, CRP, blood glucose and systolic blood pressure in some people [36,37]. Similarly, pinocembrin 7-rhamnosylglucoside, a glycosylated flavanone, although less studied than its aglycone form, may retain or modulate the antioxidant and antibacterial activities typical of flavanones. Glycosylation often influences solubility, stability and bioavailability, which may enhance the biological effect in vivo [38].
<table><caption>**Table 2**
Majority metabolites identified in hydroethanolic and methanolic extracts of stem bark analysed by UHPLC, in positive and negative mode.</caption><thead><tr><th>No peak</th><th>Rt</th><th>Name of proposed metabolite</th><th>m/z Observed</th><th>MS2(+) MS2(-)</th><th>Error (ppm)</th><th>Type of ion</th><th>Molecular Formula</th><th>Chemical family</th><th>Extracts</th><th>AL</th></tr></thead><tbody><tr><td>1</td><td>0.70</td><td>(S)-Malate</td><td>133.0143</td><td>115.0037, 133.0151, 71.0137, 89.0240</td><td>0.6</td><td>[M-H]<sup>-</sup></td><td>C<sub>4</sub>H<sub>6</sub>O<sub>5</sub></td><td>Carboxylic acid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>2</td><td>0.84</td><td>Citric acid</td><td>191.0198</td><td>191.0207, 111.0086, 87.0083, 57.0344</td><td>0.5</td><td>[M-H]<sup>-</sup></td><td>C<sub>6</sub>H<sub>8</sub>O<sub>7</sub></td><td>Carboxylic acid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>3</td><td>1.15</td><td>Disaccharides ((2Methyl-Hex)-Pen)</td><td>385.1348</td><td>385.1390, 339.1312, 207.0861, 101.0241</td><td>-1</td><td>[M+HCOO<sup>-</sup>]<sup>-</sup></td><td>C<sub>13</sub>H<sub>24</sub>O<sub>10</sub></td><td>Carbohydrate</td><td>HE; MeOH</td><td>2</td></tr><tr><td>4</td><td>2.45</td><td>Umbelliferone</td><td>163.0390</td><td>163.0399, 145.0283, 107.0495, 79.0548</td><td>0</td><td>[M + H]<sup>+</sup></td><td>C<sub>9</sub>H<sub>6</sub>O<sub>3</sub></td><td>Coumarin</td><td>HE; MeOH</td><td>2</td></tr><tr><td>5</td><td>2.84</td><td>Genipin gentiobiosiderivative</td><td>551.1974</td><td>195.0663, 177.0545, 151.0392, 107.0484</td><td>0.6</td><td>[M + H]<sup>+</sup></td><td>C<sub>23</sub>H<sub>34</sub>O<sub>15</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>6</td><td>2.90</td><td>Monotropein</td><td>389.1086</td><td>389.1089, 345.1191, 165.0560, 121.0663</td><td>-0.8</td><td>[M-H]<sup>-</sup></td><td>C<sub>16</sub>H<sub>22</sub>O<sub>11</sub></td><td>Iridoid</td><td>HE</td><td>2</td></tr><tr><td>7</td><td>2.95</td><td>Chlorogenic acid</td><td>353.0874, 355.1026</td><td>163.0397, 145.0282, 135.0431, 117.0340</td><td>-1.1, 0.6</td><td>[M-H]<sup>-</sup> [M + H]</td><td>C<sub>16</sub>H<sub>18</sub>O<sub>9</sub></td><td>Phenolic acids</td><td>HE; MeOH</td><td>2</td></tr><tr><td>8</td><td>3.05</td><td>Swertiamarin</td><td>373.1136, 375.1287</td><td>213.0754, 195.0633, 177.0547, 167.0703, 149.0596</td><td>-1.1, 0.6</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>16</sub>H<sub>22</sub>O<sub>10</sub></td><td>Iridoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>9</td><td>3.65</td><td>Feruloylquinic acid derivative</td><td>367.1030, 369.1182</td><td>329.9918, 177.0548, 145.0284, 117.0328</td><td>-1.2, 0.5</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>17</sub>H<sub>20</sub>O<sub>9</sub></td><td>Phenolic acids</td><td>MeOH</td><td>3</td></tr><tr><td>10</td><td>3.68</td><td>Pinocembrin 7-rhamnosylglucoside</td><td>565.1918</td><td>371.1313, 177.0547, 145.0284, 117.0356</td><td>0.4</td><td>[M + H]<sup>+</sup></td><td>C<sub>27</sub>H<sub>32</sub>O<sub>13</sub></td><td>Flavonoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>11</td><td>3.81</td><td>Lyoniresinol -O-glucoside derivative</td><td>627.2292</td><td>581.2191, 535.2004, 419.1461</td><td>-0.4</td><td>[M-H]<sup>-</sup></td><td>C<sub>28</sub>H<sub>38</sub>O<sub>13</sub></td><td>Lignan</td><td>HE; MeOH</td><td>3</td></tr><tr><td>12</td><td>3.82</td><td>Liriodendrin</td><td>741.2610, 760.2660</td><td>401.1599, 265.1048, 205.0864, 173.0603</td><td>0.1</td><td>[M-H]<sup>-</sup> [M+NH<sub>4</sub>]<sup>+</sup></td><td>C<sub>34</sub>H<sub>46</sub>O<sub>18</sub></td><td>Lignan</td><td>HE</td><td>2</td></tr><tr><td>13</td><td>4.17</td><td>Aspidofractine</td><td>367.2017</td><td>206.1186, 180.1008, 156.0815, 144.0815</td><td>0.2</td><td>[M + H]<sup>+</sup></td><td>C<sub>22</sub>H<sub>26</sub>N<sub>2</sub>O<sub>3</sub></td><td>Alkaloid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>14</td><td>4.29</td><td>Acetylajmaline derivatives</td><td>369.2174</td><td>369.2156, 326.1746, 325.1297, 222.1279, 158.0959, 144.0812</td><td>0.3</td><td>[M + H]<sup>+</sup></td><td>C<sub>22</sub>H<sub>28</sub>N<sub>2</sub>O<sub>3</sub></td><td>Alkaloid</td><td>HE; MeOH</td><td>3</td></tr><tr><td>15</td><td>4.46</td><td>Phellamurin</td><td>519.1862</td><td>131.0486, 103.0538, 95.0493</td><td>0.2</td><td>[M + H]<sup>+</sup></td><td>C<sub>26</sub>H<sub>30</sub>O<sub>11</sub></td><td>Flavonoid</td><td>HE; MeOH</td><td>2</td></tr><tr><td>16</td><td>4.20</td><td>di-O-caffeoylquinic acid derivative</td><td>515.1192, 517.1343</td><td>499.1245, 163.0400, 145.0285, 135.0433, 89.0388</td><td>-0.7, 0.4</td><td>[M-H]<sup>-</sup> [M + H]<sup>+</sup></td><td>C<sub>25</sub>H<sub>24</sub>O<sub>12</sub></td><td>Phenolic acids</td><td>HE; MeOH</td><td>3</td></tr></tbody></table>

Rt: Retention time; AL: Annotation Level; HE: Hydroethanolic; MeOH: Methanolic.

Dalpanin, a methoxylated flavonoid, is known for its antioxidant activities and antimicrobial potential. Although specific data remains limited, its structure suggests moderate radical activity but better metabolic stability, which may favour prolonged effects under biological conditions [39]. The presence of these bioactive compounds

suggests a potential for synergistic pharmacological effects, especially in contexts of oxidative stress, inflammation, or microbial infections, although this requires further experimental validation.
<figure><img src="image_6.png" /><figcaption>Fig. 5. Chemical structure of some metabolites identified in *Strychnos innocua* DELILE extracts.</figcaption></figure>

### 3.4.5. Polyphenolic compounds: phenolic acids

The results show that our root and stem bark extracts contain polyphenolic compounds. Analysis of the stem bark data (Fig. 4B) identified the presence of chlorogenic acid, which elutes at different retention times (Table S3). Visualisation of their MS/MS spectra confirmed that they are different isomers. Chlorogenic acids are derived from the esterification of cinnamic acids, including caffeic, ferulic and p-coumaric acids. In addition, chlorogenic and quinic acids represent the abundant polyphenolic group of plants. The polyphenolic metabolites are subdivided into several subgroups, including caffeoylquinic, p-coumaroylquinic and feruloylquinic acids [40]. Caffeoylquinic acids (CQAs), esters of caffeic acid and quinic acid, are among the important polyphenols found in the plant families *Asteraceae* and *Lamiaceae* [41] and are one of the main components of coffee [42]. 3-Caffeoylquinic acid (3-ACQ or neochlorogenic acid) is present in several plants, such as coffee, tomatoes, artichokes, potatoes, apples and fresh stone fruit (peaches and plums) [41,42]. 5-ACQ, also known as chlorogenic acid, is the most abundant isomer of caffeoylquinic acid derivatives (3, 4 and 5-ACQ) and has numerous beneficial effects on human health [41,43]. In vitro, caffeoylquinic acids, including 3-ACQ, have α-glucosidase and DPP-4 inhibitory activities [44]. Phenolic acids are recognised for their numerous antioxidant properties and their beneficial effects on health. In particular, they protect against disorders linked to oxidative stress, such as cardiovascular diseases and various cancers. They also have

antimicrobial and hypoglycaemic activities [45].

### 3.4.6. Alkaloids

In addition to the other families of compounds mentioned above, the presence of alkaloids was detected in extracts of *Strychnos innocua* root bark (17%, Fig. 4A) and stem bark (12%, Fig. 4B). Several bioactive alkaloids were detected, including cinchonine (Table 1), aspidofractin, ajmaline derivatives (Table 2) and other related structures. Although these compounds are traditionally associated with other botanical genera, their identification in *S. innocua* suggests either a biosynthetic convergence within the Loganiaceae family, or a broader chemical diversity than that described to date in the genus *Strychnos*. Indeed, several species in the genus, such as *S. nux-vomica*, *S. icaja*, *S. nitida* and *S. spinosa*, are well documented for the production of major bisindolic or monoindolic alkaloids, including strychnine, brucine, sungucine and vomicine, whose biological activities are widely reported in the literature [46].

The alkaloids identified in *S. innocua* are also of significant pharmacological interest. Cinchonine is one of the *Cinchona* alkaloids, extracted commercially from the Peruvian bark of *Cinchona officinalis* L. (family: *Rubiaceae*). It has historically been recognised as an antimalarial agent. It also has a wide range of other biological properties, including anti-cancer, anti-obesity, anti-inflammatory, anti-parasitic, antimicrobial, anti-platelet aggregation and anti-differentiation of
osteoclasts [47]. Aspidofractin, a monoterpenoid-indolic alkaloid of the aspidosperma type, is characteristic of several African medicinal plants. Despite its lack of documentation, it is part of a family of compounds that show anti-tumour, anti-microbial and sometimes neuroactive activities. The presence of this alkaloid suggests cytotoxic and topoisomerase inhibitory potential, as do some similar alkaloids [48]. Ajmaline derivatives are well established for their antiarrhythmic properties. Ajmaline acts by blocking cardiac sodium channels, making it a reference treatment for Brugada syndrome and other cardiac rhythm disorders [49]. Acetylation can modify the lipophilicity and bioavailability of these compounds, potentially influencing their pharmacological activity.

### 3.4.7. Synthesis and pharmacological relevance

Comparative metabolite profiling of *Strychnos innocua* root and stem bark revealed a wide diversity of secondary metabolites, including iridoids, secoiridoids, alkaloids, flavonoids and phenolic acids. Iridoids were particularly abundant in the root barks, while polyphenolic compounds predominated in the stem barks. This highlights the complementary chemical profiles of the two organs.

Beyond their chemical significance, these metabolites are renowned for their antioxidant, anti-inflammatory and antimicrobial properties, which align with the species' pharmacological potential. Notably, the chemical richness of *S. innocua* provides a strong basis for its traditional medicinal use, particularly in the management of sickle cell disease. This relevance is further reinforced by our previous work, in which the anti-sickling activity of *S. innocua* extracts was demonstrated experimentally [8]. Thus, the chemical diversity characterised in the present study offers a plausible explanation for the biological activities reported in traditional medicine and in our own experimental findings.

Future studies should aim to isolate additional bioactive compounds, clarify their mechanisms of action and evaluate their combined effects, in order to determine whether they act synergistically or additively. Such an approach would bridge the gap between phytochemical composition and pharmacological efficacy, supporting the development of *S. innocua* as a potential source of therapeutic agents.

Although the present study focuses on the chemical characterization and isolation of bioactive compounds from *Strychnos innocua* Delile, the metabolites identified may have potential pharmacological effects relevant to various pathophysiological processes, including mitochondrial function. Detailed mechanistic studies are beyond the scope of this work; however, readers interested in mitochondrial targets and disease-related mechanisms can refer to recent studies for further insights [50–52].

### 3.5. Compounds isolated from root barks

Compound C1 was isolated from the methanolic extract. This compound is a light-yellow powder, soluble in methanol and water. Negative mode electrospray ionisation mass spectrometry (HRESIMS) analysis of C1 gave two pseudo molecular ion peaks corresponding to *m/z* 393.0963 (error *m/z* = -0.4 ppm) [M - Cl]⁻ and *m/z* 357.1182 (error *m/z* = 1.1 ppm) [M - H]⁻, suggesting a molecular weight of 358 and compatible with the molecular formula C₁₆H₂₂O₉ containing 6 degrees of unsaturation (Figure S7). Fragmentation gave an ion observed at *m/z* 195.0663 [M-H⁻]⁻ 162 corresponding to the loss of a hexosyl group. Analysis of the 1H NMR spectrum (Figure S8) of compound C1 recorded in deuterated methanol (MeOD) shows several types of protons, namely: a doublet at 7.61 ppm (*J* = 2.75 Hz), which is characteristic of the C-3 proton of the dihydropyran ring of the iridoids; signals between 3.19 and 3.90 ppm, characteristic of osidic protons and a doublet signal (*J* = 7.75 Hz) resonating at 4.70 ppm, characteristic of the anomeric H-1' proton. The NMR of the carbon (Figure S9) and the DEPT 135 spectrum (Figure S10) show 4 methylene groups (CH₂), 10 methines and 2 quaternary carbons. The chemical shifts and signal assignments are shown in Table 3. By comparing our 1D NMR data with those of Inouye *et al*.,

<table><caption>**Table 3**
1H NMR and 13C NMR data for compounds C1 and C2 in CD₃OD.</caption><thead><tr><th rowspan="2">N°</th><th colspan="2">C1</th><th colspan="2">C2</th></tr><tr><th>δ<sub>C</sub> (ppm)</th><th>δ<sub>H</sub> (ppm)</th><th>δ<sub>C</sub> (ppm)</th><th>δ<sub>H</sub> (ppm)</th></tr></thead><tbody><tr><td>1</td><td>96.58</td><td>5.57 m</td><td>97.65</td><td>5.48 d (J = 1.9 Hz)</td></tr><tr><td>3</td><td>152.55</td><td>7.61 d (2.75 Hz)</td><td>148.81</td><td>7.36 d (J = 2.5 Hz)</td></tr><tr><td>4</td><td>104.63</td><td></td><td>108.95</td><td></td></tr><tr><td>5</td><td>27.05</td><td>3.17 m</td><td>28.83</td><td>3.04 dtd (J = 12.8 ; 4.8 ; 2.7 Hz)</td></tr><tr><td>6</td><td>24.43</td><td>1.73 m<br>1.78 m</td><td>25.95</td><td>1.58 m<br>1.75 m</td></tr><tr><td>7</td><td>68.30</td><td>4.38 td (11.95 ; 2.65 Hz)<br>4.48 dq (11.3 ; 2.15 Hz)</td><td>62.65</td><td>3.67 m<br>3.90 m</td></tr><tr><td>8</td><td>31.94</td><td>5.56 - 5.59 m</td><td>134.10</td><td>5.57</td></tr><tr><td>9</td><td>42.44</td><td>2.72 m</td><td>44.59</td><td>2.67 ddd (J = 9.7 ; 5.5 ; 1.8 Hz)</td></tr><tr><td>10</td><td>119.41</td><td>5.29 m<br>5.32 m</td><td>120.27</td><td>5.12-5.30 m</td></tr><tr><td>11</td><td>167.11</td><td></td><td>168.39</td><td></td></tr><tr><td>1'</td><td>98.31</td><td>4.70 d (7.75 Hz)</td><td>99.64</td><td>4.67 d (J = 7.9 Hz)</td></tr><tr><td>2'</td><td>73.33</td><td>3.19 m</td><td>74.74</td><td>3.07 m</td></tr><tr><td>3'</td><td>76.49</td><td>3.39</td><td>77.94</td><td>3.27 m</td></tr><tr><td>4'</td><td>70.13</td><td>3.29 m</td><td>71.53</td><td>3.20 m</td></tr><tr><td>5'</td><td>76.99</td><td>3.28 m</td><td>78.30</td><td>3.19 m</td></tr><tr><td>6'</td><td>61.27</td><td>3.68 dd (12 ; 5.9 Hz)<br>3.90 dd (11.85 ; 2.3 Hz)</td><td>41.74</td><td>3.24</td></tr></tbody></table>

(1976), we can conclude that C1 is the sweroside Fig. 6A [53].

Compound C2 was isolated from the methanolic extract of *S. innocua* root bark. It is a white powder, soluble in methanol and water. The mass spectrum (Figure S11) shows two pseudo molecular ion peaks corresponding to *m/z* 392.1113 (error *m/z* = 0.1 ppm) [M - Cl]⁻ and *m/z* 356.1355 (error *m/z* = 1 ppm) [M - H]⁻, suggesting a molecular weight of 357. The odd molecular weight implies an odd number of nitrogen atoms, which is compatible with the molecular formula C₁₆H₂₃NO₈ containing 6 degrees of unsaturation. The ¹H NMR spectrum (Figure S12) of compound C2 recorded in deuterated methanol shows several signals characteristic of a secoiridoid skeleton and a sugar, which can be identified through: a doublet at 7.25 ppm (*J* = 2.5 Hz), which is characteristic of the C-3 proton of the dihydropyran ring of secoiridoid; a doublet (*J* = 7.9 Hz) resonating at 4.67 ppm characteristic of the anomeric H-1' proton in the β position; signals between 3.07 and 3.30 ppm, characteristic of osidic protons; a doublet at 5.48 (*J* = 2 Hz) ppm characteristic of the H-1 proton; a resonant doublet at 2.67 ppm attributable to the H-9 proton; signals in the form of a multiplet at 3.67 and 3.90 ppm corresponding to the H-7 proton. The chemical shift of these latter protons suggests amidation of the C-7 carbon that carries them. The NMR of the carbon (Figure S13) and the DEPT 135 spectrum (Figure S14) indicate 4 methylene groups (CH₂), 10 methines and 2 quaternary carbons. The chemical shifts and signal assignments are shown in Table 3.

Analysis of the COSY H-H spectrum (Figure S15) reveals several correlations: H-1/H-9; H-9/H-5 and H-9/H-8; H-3/H-5; H-6/H-7; H-1'/H-2'. The HMBC spectrum (Figure S16) confirms the secoiridoid skeleton by the presence of the following correlations: H-3 shows correlation spots with carbons C-1 (97.65 ppm), C-4 (108.95 ppm) and C-5 (28.83 ppm); H-1 shows correlation spots with carbons C-5 (28.83 ppm), C-3 (148.81 ppm) and the anomeric carbon C-1' (99.64 ppm) Fig. 6B This implies that β-D-glucose is bound to the iridoid backbone at the C-1 position; H-10 shows correlation spots with carbons C-8 (134.10 ppm) and C-9 (44.59 ppm). Analysis of our 1D and 2D NMR data with those in the literature [54] suggests that C2 is the bakankoside Fig. 6B

Bakankoside and Sweroside were identified in our root bark extracts and are among the major compounds. These two compounds were isolated from the methanolic extract of the root using a combination of chromatographic and analytical techniques. Sweroside was first isolated
<figure><img src="image_7.png" /><figcaption>Fig. 6. A) Structure of compounds C1; B) HMBC correlation of the secoiridoid skeleton and structure of C2.</figcaption></figure>

from the plant *Swertia japonica* in 1966 in Japan [53]. It has been the subject of several biological studies. It is known to have anti-inflammatory, hepatoprotective, antidiabetic, antifungal, anti-tumour, antiproliferative and vasorelaxant activity [55]. In addition, this molecule has already been isolated from other plants and even from certain species of *Strychnos*, namely *S. nux-blanda* [19], *S. spinosa* [21] and *S. axillaris* [56]. Bakankoside is a monoterpene glycosidic alkaloid which was first isolated in 1907 from *Strychnos vacacoua* [57]. It is one of the rare secoiridoids to possess a nitrogen atom. To our knowledge, these two molecules were identified for the first time in *Strychnos innocua*.

## 4. Conclusion

This study provides the first comprehensive chemical characterization of the root and stem barks of *Strychnos innocua* using UHPLC-MS/MS and semi-preparative HPLC. A total of 150 and 118 metabolites were annotated in the root and stem barks, respectively, with two compounds identified at level 1. The chemical profiles revealed a predominance of iridoids in the root bark, and polyphenolic compounds in the stem bark. Two compounds, sweroside (iridoid) and bakankoside (monoterpene alkaloid), were successfully isolated and structurally characterised.

The richness and diversity of secondary metabolites in *S. innocua* not only underscores its potential as a source of bioactive molecules but also supports its traditional use in the management of sickle cell disease, as previously confirmed by our anti-sickling studies. Future work should focus on the purification of additional metabolites and the evaluation of their biological activities, both individually and in combination; in order to validate potential synergistic effects and clarify their pharmacological relevance.

## Financial

No funding was received.

## Data availability

All data analysed during this study are included in this published article and its supplementary information files.

## CRediT authorship contribution statement

**Mawaté Florance Brukum:** Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation.
**Isabelle Schmitz:** Writing – review & editing, Methodology, Formal analysis, Data curation.
**Carlos Afonso:** Validation, Investigation,

**Conceptualization.** **Abdelhakim Elomri:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Conceptualization.
**Kafui Kpegba:** Writing – review & editing, Supervision, Project administration, Conceptualization.
**Kodjo Selom Evenamede:** Validation, Investigation.
**Amegnona Agbonon:** Writing – review & editing, Supervision, Project administration.
**Oudjaniyobi Simalou:** Investigation.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Reports a relationship with that includes:. Has patent pending to. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

This work was made possible in part by a grant from the French government awarded by Togo's Department of Cooperation and Cultural Action. This work has also been partially supported by University of Rouen-Normandie, INSA Rouen-Normandie, the Centre National de la Recherche Scientifique (CNRS), Région Normandie, European Regional Development Fund (ERDF), Labex SynOrg (ANR-11-LABX-0029), Carnot Institute I2C, and by the graduate school for research XL-Chem (ANR-18-EURE-0020 XL CHEM).

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.nexres.2025.101217.

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