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. 2025 Dec 5;23(1):e02467. doi: 10.1002/cbdv.202502467

Analysis of Phytochemicals and Functional Groups From Aqueous Extracts of Malawian Vernonia amygdalina Del. for Potential Applications

Bonface Mwamatope 1,2,3,✉, Elijah Malinga Menard Wanda 2,3, Ibrahim Chikowe 4,5, Naomi Chikwana 1, Fednand Paul Wanjala 2,3, Lesten Eliez Chisomo Chatepa 1, John Finias Kamanula 2,3
PMCID: PMC13420621  PMID: 41348477

ABSTRACT

Vernonia amygdalina Del., a plant recognised for its medicinal properties, is widely available in Malawi, but remains underutilised. The study aimed to analyse phytochemicals and their functional groups in the aqueous extracts of V. amygdalina Del. leaves. The functional groups of the compounds were determined using Fourier‐transform infrared spectroscopy (FT‐IR). Phenolic compounds were quantified using high‐performance liquid chromatography‐ultraviolet visible assay. Gas chromatography mass spectrometry was used to identify volatile phytoconstituents. The FT‐IR spectrum revealed characteristic absorption bands corresponding to C═C, C═O, C═O, ─OH and ─NH active functional groups. The phenolic compounds identified included gallic acid, sinapic acid, vanillic acid, ferulic acid, rutin and quercetin, whose concentrations varied significantly (p < 0.05). Volatile compounds (21) were observed with dl‐α‐Tocopherol as a major compound, followed by β‐sitosterol, ethyl iso‐allocholate, 9,12,15‐octadecatrienoic acid, 2,3‐dihydroxypropyl ester, (Z,Z,Z)‐ and stigmasterol. The aqueous extracts of V. amygdalina Del. leaves contained phytochemicals with active functional groups that can act as reducing and capping agents, thereby demonstrating potential applications in green chemistry. Based on these findings, we recommend further studies using this extract to actually synthesise silver and zinc oxide nanoparticles and characterise them using spectroscopic techniques for use in promoting sustainable farming practices.

Keywords: functional groups, green chemistry, HPLC‐UV‐Vis, nanoparticles and phytochemicals

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1. Introduction

Vernonia amygdalina Del., also known as bitter leaf and Tsoyo or Futsa in Malawi, is one of the commonly available plant species. It has some medicinal properties, but is underutilised in Malawi [1, 2]. It has been reported to be effective in the management of diseases in different countries, including Malawi, Nigeria, Ethiopia, Tanzania, Rwanda, Guinea, Benin and Sierra Leone [3, 4]. Tembo et al. [5] observed that the Malawian V. amygdalina Del. is effective in the management of arthropods in legume crops. In another study, Nyirenda et al. investigated plants with known pesticide and medicinal use in crops, livestock, and households in Lilongwe, Malawi. It was documented that farmers use leaves of V. amygdalina Del. to manage diseases in animal farming [6]. Peasant farming is important because it provides food and also additional income to less privileged smallholder farmers [7]. However, the high incidence of diseases is one of the major constraints to smallholder farmers in developing countries, including Malawi [8]. Inability to access conventional drugs leads poor farmers to rely on herbal remedies [9]. For example, ethno‐veterinary medicine is widely used by most rural farmers, and they claim that these practices are effective [10, 11]. Application of Malawian herbal medicines has a long history of use in treating various diseases in Malawi [12, 13, 14, 15, 16].

Various methods to improve herbal medicines have been proposed, including the combination of herbal medicines with nanotechnology. V. amygdalina Del. contains bioactive compounds such as flavonoids and sesquiterpene lactones [3, 17], alkaloids, polyphenols, proteins, sugars, and terpenoids [18, 19], which are believed to have an important role in the green synthesis and stabilisation of metal nanoparticles (NPs) [20]. These compounds act as reducing and capping agents due to the presence of active functional groups [21]. Green chemistry in nanotechnology is an environmentally friendly approach that offers a sustainable alternative to conventional chemical and mechanical methods. V. amygdalina Del. has been used to synthesise metal nanoparticles such as silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) in various countries [22, –, 25]. However, no information is available on the use of synthesised AGNPs and ZnONPs using Malawian V. amygdalina Del.

Despite available information on phytochemicals in V. amygdalina Del. from other countries, different environmental conditions, leaf maturity stage, seasonal variations, genetic predisposition of the plant, and reactions against seasonal diseases or pests may contribute to the variations in the availability or concentration of secondary metabolites in the plant species [26–, 29], hence the need for further studies. The phytochemical profile of Malawian V. amygdalina Del., particularly their aqueous extracts, is limited [30] and remains uncharacterised for potential application in the synthesis of metal NPs. Therefore, the study aimed at analysing phytochemicals and their functional groups in the aqueous extracts of V. amygdalina Del., leaves, which are crucial for understanding their chemical reactivity and potential uses in green nanotechnology. The results will help to bridge the gap of limited information on the chemical profiles of phytochemicals in the aqueous extract of the Malawian V. amygdalina Del. It will also help to predict the reaction mechanisms of bioactive compounds involved in the green synthesis of the NPs that could provide eco‐friendly solutions for the management of diseases, such as potato bright, Newcastle virus, and infectious coryza in the promotion of sustainable farming practices.

2. Results and Discussion

2.1. Functional Group Profiling

Fourier‐transform infrared spectroscopy (FT‐IR) is used to identify the types of functional groups present in the phytochemicals that are capable of stabilising NPs, preventing agglomeration, and creating their capping in an aqueous solution [31]. The chemical bonds of a compound are determined by interpreting the molecular fingerprint of the infrared absorption spectrum, as shown in Figure 1 [32].

FIGURE 1.

FIGURE 1

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Fourier‐transform infrared spectroscopy (FT‐IR) absorption spectrum of the aqueous extract of V. amygdalina Del.

The FT‐IR spectrum of the aqueous extract of V. amygdalina Del. leaves in Figure 1 showed a broad absorption band from 3620 to 3200 cm−1, which was attributed to the overlap stretching of ─OH and ─NH functional groups. The peak of absorption of 2848 cm−1 indicated that the carboxylic ─OH group was stretched due to a hydrogen bond between the solvent (water) and bioactive compounds such as phenolic compounds and organic acids [33]. The presence of phenolic compounds is further supported by the aromatic C═C stretching band detected at 1643 cm−1. The existence of organic acids is also supported by the 1763 and 1241 cm−1 absorption bands of the C═O and C─O stretching vibrations, respectively. Furthermore, the stretching vibrations of ethers produced a peak of 1012 cm−1. The absorption bands at 2359 and 2109 cm−1 implied the presence of conjugated compounds with C═C and C≡C groups, respectively. Therefore, the aqueous extract of V. amygdalina Del. contained phytochemicals with functional groups that can play an important role as reducing and capping agents in the green synthesis of metal and metal oxide NPs through redox reactions (Figure 2) [32]. Some water‐soluble molecules can reduce metal ions by converting the C─O group to the ─C─O and oxidising the aldehyde groups of the molecules into carboxylic acids [34]. Furthermore, when metal salts are added to plant extracts, metal ions bind to the functional groups of biomolecules such as ─OH, thereby reducing captured metal ions into metal NPs through redox reactions [35, 36]. For example, phenolic compounds may donate electrons or hydrogen atoms with the constraints of the carbon valency and the chelating action of the adjacent hydroxyl groups in the synthesis of AgNPs. The phenolic compounds are oxidised to ketone, while the silver ions are reduced to Ag atoms that combine into AgNPs, followed by capping, as shown in Figure 2 [37].

FIGURE 2.

FIGURE 2

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Plausible reaction mechanism of the silver ion reduction and cupping by phytochemicals.

Genc et al. [38] observed that some peaks of the plant extract were modified or disappeared, while new peaks appeared in the FT‐IR spectrum of the AgNPs [38]. This implied involvement of the functional groups in the synthesis of the AgNPs. In another study of ZnONPs synthesis, a characteristic hydroxyl (‐OH) stretching absorption band was identified in the biosynthesised ZnONPs, which indicated the formation of zinc complexes with compounds in plants [39]. Some of the values of FT‐IR in the aqueous extract of V. amygdalina Del. correlate with other findings that have been reported before [40].

2.2. Phenolic Compounds Identification and Quantification

Fingerprints of standard compounds can serve as a reference point for analysing and comparing unknown compounds using high‐performance liquid chromatography (HPLC) in extracts [41]. A chromatogram of six standard compounds: gallic acid (3,4,5‐trihydroxybenzoic acid); sinapic acid (4‐hydroxy‐3,5‐dimethoxycinnamic acid); vanillic acid (4‐hydroxy‐3‐methoxybenzoic acid); rutin (3′,4′,5,7‐Tetrahydroxy‐3‐[α‐L‐rhamnopyranosyl‐(1→6)‐β‐D glucopyranosyloxy]flavone); ferulic acid (4‐hydroxy‐3‐methoxycinnamic acid) and quercetin (3,5,7,3',4'‐pentahydroxyflavone) that were used for peak identification is presented in Figure 3a.

FIGURE 3a.

FIGURE 3a

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High‐performance liquid chromatography‐ultraviolet visible (HPLC‐UV‐Vis) chromatogram of standards: Gallic acid (1); sinapic acid (2), vanillic acid (3); rutin (4), ferulic acid (5) and quercetin (6).

A chromatogram of a sample after using the optimised method is shown in Figure 3b. The chromatogram of the aqueous extract shows more peaks of the compounds that have not been identified, implying the presence of more phytochemicals than available standards.

FIGURE 3b.

FIGURE 3b

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High‐performance liquid chromatography‐ultraviolet visible (HPLC‐UV‐Vis) chromatogram of compounds in the sample: Gallic acid (peak number 3); sinapic acid (peak number 7), vanillic acid (peak number 9); rutin (peak number 10), ferulic acid (peak number 11) and quercetin (peak number 13).

The polyphenols: gallic acid, sinapic acid, vanillic acid, rutin, ferulic acid and quercetin were found in aqueous extracts of V amygdalina Del. (Table 1). The concentrations of the phenolic compounds in the samples ranged significantly from 1.30 ± 0.01 to 27.6 ± 0.10 µg/mL (p < 0.05) (Table 1). The highest concentration was for 4‐hydroxy‐3‐methoxycinnamic acid (ferulic acid), while the lowest was for 3,4,5‐trihydroxybenzoic acid (gallic acid). The calibration curves for all reference compounds are linear (r2 ≥ 0.9786) (Table 1). The recovery of individual compounds after spiking ranged from 87% quercetin (3,5,7,3',4'‐pentahydroxyflavone) to 101% sinapic acid (4‐hydroxy‐3,5‐dimethoxycinnamic acid) (Table 1). The creation, optimisation and validation of HPLC methods are critical to the production of accurate and consistent results [42].

TABLE 1.

Phenolic compounds calibration parameters and concentrations.

Compound RT LOD LOQ Recovery Linearity Concentration
(min) (µg/mL) (µg/mL) (%) (r2) (µg/mL)
Gallic acid 3.7 0.01 0.02 89 0.9921 1.30 ± 0.01f
Sinapic acid 12.2 0.03 0.25 101 0.9886 17.2 ± 0.30c
Vanillic acid 12.8 0.01 0.11 93 1.000 7.60 ± 0.30e
Rutin 13.3 0.04 0.21 90 0.9997 26.8 ± 1.1b
Ferulic acid 13.6 0.03 0.33 88 0.9786 27.6 ± 0.10a
Quercetin 14.9 0.01 0.91 87 0.9988 12.5 ± 0.40d
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Abbreviations: Concentrations are means of triplicate samples (n = 3, ± standard deviation). Mean values with different letters indicate significant differences (Tukey's test, p < 0.05); LOD, limit of detection; LOQ, limit of quantification; RT, retention time.

The structures of identified compounds in aqueous extracts of V. amygdalina Del. using HPLC‐ultraviolet visible (HPLC– UV‐Vis) are presented in Figure 4. The functional groups of the identified compounds correlate with the results of FT‐IR (Figure 1). Sinapic and ferulic acids contain the hydroxy group at the para position and possess a C═C double bond between the phenyl ring and the carboxylic acid group. The para‐hydroxy substitution makes these cinnamic acid derivatives have the highest activity. This is because the C═C bond shields the ring from the electron‐withdrawing effect of the carboxyl group [43].

FIGURE 4.

FIGURE 4

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The structures of observed phenolic compounds.

Quercetin is a polyphenol that contains a 3‐hydroxy group and a C═C double bond at C2 and C3 in the O‐containing ring. The 3‐hydroxyl group and conjugated system enable quercetin to exhibit high activity and good resonance stabilisation of radicals [43]. Apart from transferring electrons to ions or radicals, quercetin is an excellent antioxidant because it can donate hydrogen atoms from its aromatic hydroxyl groups to free radicals [44].

Kubavat et al. observed that rutin, a natural flavonoid‐based sugar, can be used as a reducing agent in the synthesis of AgNPs due to its antioxidant activity potential [45]. Quercetin, gallic acid, and ferulic acid were reported in the synthesis of AgNPs and selenium NPs [46, 47], while sinapic acid has been reported for use in the synthesis of stable copper oxide NPs [48]. In another study, vanillic acid, which is an active dietary phenolic compound and an oxidised form of vanillin, was used in the synthesis of a nanocomposite comprising chitosan and AgNPs [49].

Plants have higher levels of the phenolic compounds, known as antioxidants, which can inhibit or prevent the oxidation of oxidizable molecules by scavenging free radicals, reactive oxygen, nitrogen and sulphur species (ROS/RNS/RSS) [50]. It is concluded that these antioxidants are also responsible for the biosynthesis of metal or metal oxide NPs, such as AgNPs and ZnONPs, because they can reduce metal ions and stabilise the produced NPs [51]. In the green synthesis of ZnONPs, phenolic compounds are believed to lower the valence of zinc ions to zero by donating electrons from the hydroxyl groups [52, 53, 54]. This is followed by the addition of oxide to metallic zinc by calcination or oxidation to form ZnO nuclei [51, 53, 54]. Another study suggested that the zinc (II) ions were reduced by the active compounds in plants, which reacted with dissolved oxygen in the solution and formed ZnO nuclei [51, 55, 56]. The presence of phenolic compounds such as gallic acid, rutin, and quercetin in aqueous extracts of V. amygdalina Del. is consistent with another study [57].

2.3. Volatile Phytochemicals Identification

Comprehensive compound identification using gas chromatography mass spectrometry (GC‐MS) analysis identified 21 compounds, including major components like dl‐α‐Tocopherol, β‐sitosterol and Ethyl iso‐allocholate. The total ion gas chromatogram is shown in Figure 5, and all identified compounds are listed in Table 2 with their retention times (RT), concentration (% peak area), molecular formula, and molecular weight.

FIGURE 5.

FIGURE 5

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Total ion gas chromatogram of aqueous leaf extract of V. amygdalina Del.

TABLE 2.

Compounds identified by gas chromatography‐mass spectrometry (GC‐MS) in the aqueous extract of V. amygdalina Del.

S/N

RT

(min)

 Abundance (%) Compound name Compound class Molecular formula Molecular weight (g/mol)
1 12.445 0.18 Cyclotetrasiloxane, octamethyl‐ Organosilicon siloxane C8H24O4Si4 296.61
2 19.072 0.23 3‐Octen‐2‐ol, (Z)‐ Fatty alcohol C8H16O 128.21
3

21.453

 0.12 1‐Methylene‐2b‐hydroxymethyl‐3,3‐dimethyl‐4b‐(3 methylbut‐2‐enyl)‐cyclohexane Sesquiterpene alcohol C15H26O 222.37
4 22.323 0.60 β‐D‐Glucopyranose, 1,6‐anhydro‐

Anhydrosuga

 C 6H10O5 162.14
5 22.862 0.11 Preg‐4‐en‐3‐one, 17α‐hydroxy‐17β‐cyano‐ Progestin steroid C 20H27NO2 313.4
6 23.297 0.26 Dodecanoic acid Fatty acid C12H24O2 200.32
7 23.959 0.15 (2E)‐1‐Methoxy‐3,7‐dimethylocta‐2,6‐diene Ether C 11H20O 168.28
8 25.740 0.30 1‐Undecene, 8‐methyl‐ Alkene C 12H24 168.32
9 27.314 4.33 Bicyclo[3.1.1]heptane, 2,6,6‐trimethyl Bicyclic terpenoid C 10H18 138.25
10 27.625 2.94 Bicyclo[2.2.1]heptane, 1,3,3‐trimethyl Bicyclic terpenoid C 10H18 138.25
11 29.033 2.77 n‐Hexadecanoic acid Fatty acid C 16H32O2 256.42
12 29.882 0.17 (2E)‐1‐Methoxy‐3,7‐dimethylocta‐2,6‐diene Ether C 11H20O 168.28
13 31.249 6.56 9,12,15 octadecatrienoic acid, 2,3‐dihydroxypropyl ester, (Z,Z,Z)‐ Organic ester C21H36O4 352.5
14 32.326 4.12 Octadecane Alkane C18H38 254.5
15 35.184 49.37 dl‐α‐Tocopherol Phenolic compound C29H50O2 430.7
16 37.400 8.00 Ethyl iso‐allocholate Organic ester C 26H44O5 436.6
17 37.897 2.08 Hexadecanoic acid, 2‐hydroxy‐, methyl ester Organic ester C 17H34O3 286.4
18 38.373 4.02 Stigmasterol Steroids C29H48O 412.7
19 39.554 0.92 8‐Dodecen‐1‐ol, acetate, (Z)‐ Acetate ester C 14H26O2 226.35
20 40.299 8.81 β‐Sitosterol Steroids C 29H50O 414.7
21 41.355 3.96 Lanosterol Steroids C 30H50O 426.7
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The results showed that dl‐α‐Tocopherol (49.37%) is the main component, followed by β‐sitosterol (8.81%) and Ethyl iso‐allocholate (8.00%). Of the 21 compounds identified, 17 contained oxygen. Compounds containing functional groups such as ─OH, ─OCH3 and C═O are reported to reduce and easily bind metal ions and convert them into metal NPs [58]. The presence of compounds of different classes, such as alcohols, organic acids, phenolic compounds, alkenes, ketones and ethers, correlates with FT‐IR results (Figure 1).

The structures of some compounds of the interest, including dl‐α‐Tocopherol (49.37%; RT 35.184 min), β‐sitosterol (8.81%; RT 40.299 min), Ethyl iso‐allocholate (8.00%; RT 37.400 min), 9,12,15 octadecatrienoic acid, 2,3‐dihydroxypropyl ester, (Z,Z,Z)‐ (6.56%; RT 31.249 min), Stigmasterol (4.02%; RT 38.373 min), and Dodecanoic acid (0.26%; RT 23.297 min), are shown in Figure 6a–f. α‐tocopherol is the major vitamin E group of lipophilic compounds that are synthesised by plants. Tocopherols have concentration variations under abiotic stress, suggesting their essential function as antioxidants and as an indicator of plant stress tolerance [59]. As an antioxidant, α‐tocopherol deactivates photosynthesis‐derived ROS and scavenges lipid peroxyl radicals in thylakoid membranes to prevent the spread of lipid peroxidation [60]. The mass spectrum and chemical structure of dl‐α‐Tocopherol (Figure 6a) show that tocopherol is an organic compound composed of methylated phenol. The phenolic hydroxyl group of the chromanol ring allows the compound to act as an antioxidant and can reduce metal ions, as summarised in the graphic abstract [61]. β‐sitosterol (Figure 6b) and stigmasterol (Figure 6c) are stigmast‐5‐ene substituted by a beta‐hydroxy group at position C‐3 of the structure; hence β‐sitosterol is also a 3beta‐hydroxy‐Delta (5)‐steroid with 29 carbons. In stigmasterol, there are unsaturated bonds in positions 5‐6 of the B ring and position 22‐23 of the alkyl substituent. The compounds are derivatives of stigmastane, a member of phytosterols that have anti‐inflammatory, anti‐tumor and anti‐fibrotic effects [62]. β‐sitosterol has been reported to play a role as an inhibitor of sterol methyltransferase, an anticholesteremic drug, and an antioxidant due to its structure [63].  In another study, Lucas‐Gomez and others reported that phytosterols contained in Myrtillocactus geometrizans were responsible for reducing and stabilising AgNPs below 5 nm [64]. The presence of β‐sitosterol in extracts of Vernonia amygdalina Del. is consistent with another study done by Garba et al. [65].

FIGURE 6.

FIGURE 6

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Mass spectrum and structure of compounds.

Mass spectrum and chemical structure of ethyl iso‐allocholate (Figure 6d), a steroidal compound, show that it contains functional groups such as hydroxyl. In another study, the aqueous extracts of V. amygdalina Del. significantly reduced glutathione level, superoxide dismutase, and catalase activities, with concomitant depletion of malondialdehyde level due to the presence of bioactive compounds including ethyl iso‐allocholate [66]. The presence of hydroxyl groups on ethyl iso‐allocholate (Figure 6d), 9,12,15‐octadecatrienoic acid, (Z)‐2,3‐dihydroxypropyl ester (Figure 6e) and dodecanoic acid (Figure 6f) increases the ability of these compounds to be natural antioxidants and reducing agents [43, 66, 67]. For example, Ramana et al. reported that dodecanoic acid, also known as lauric acid, reduced and capped AgNPs without any aggregation [68].

3. Conclusion

The aqueous extracts of V. amygdalina Del. leaves contained phenolic compounds and other phytochemicals with functional groups such as ‐OH, ─COOH, C═C, C─O, and ─C─O. The HPLC‐UV‐Vis chromatogram of the sample revealed more peaks of the compounds, implying the presence of more phytochemicals. A comprehensive identification of compounds using GC‐MS analysis identified 21 compounds, including major components such as dl‐α‐Tocopherol, β‐sitosterol, and Ethyl iso‐allocholate. The presence of phenolic compounds and other phytochemicals suggests that the aqueous extracts of V. amygdalina Del. leaves can serve as reducing and capping agents, hence they can be applied in the green synthesis of metal NPs. Green nanotechnology is an environmentally friendly approach that is a sustainable alternative to conventional chemical and mechanical synthesis methods. Based on these results, we recommend further studies using this extract to actually synthesise AgNPs and ZnONPs that could provide eco‐friendly solutions for the management of diseases, such as potato bright, Newcastle virus, and infectious coryza in the promotion of sustainable farming practices. In addition, we recommend characterisation of the NPs to be synthesised using spectroscopic techniques such as UV‐Vis spectroscopy, dynamic light scattering, scanning electron microscopy—energy‐dispersive X‐ray spectroscopy, transmission electron microscopy, X‐ray diffraction and atomic force microscopy.

4. Materials and Methods

4.1. Chemical Reagents

HPLC grade reagents (acetic acid and methanol) and the standards of purity equal or greater than 99.5% (gallic acid, sinapic acid, vanillic acid, ferulic acid, rutin and quercetin) were purchased from Sigma‐Aldrich Chemical Co. (USA). All other chemicals were of analytical grade.

4.2. Plant Materials Preparation and Extraction

Leaves of V. amygdalina Del. were collected randomly from the Bunda forest in the district of Lilongwe, central region of Malawi. The samples were authenticated by a taxonomist from the National Herbarium and Botanic Gardens of Malawi (NHBG) in Lilongwe. The plant sample was assigned a voucher number (MLW‐BM/LL‐LL‐001) and deposited in the NHBG. The scientific name has been reviewed and verified in the World Flora Online Plant List at https://wfoplantlist.org/. The leaves were dried for 21 days under the shed and crushed into fine powder with a laboratory mill (Arthur H. Tomas Co., Philadelphia, USA). Extraction of phytochemicals was achieved as described earlier with some modifications [69]. Briefly, the ground sample (5 g) was soaked in 100 mL of deionised water and heated in a water bath for 1 h at 70°C while intermittently stirring. The extract was then filtered through filter paper No.1, freeze‐dried, and the final concentration was 5 mg/mL of the original extract's lyophilised powder. Subsequently, it was stored at 4°C for further analysis.

4.3. Functional Group Profiling

Functional groups of compounds present in aqueous extracts of V. amygdalina Del. leaves were determined by the Fourier‐transform Infrared spectrophotometer (Shimadzu FT‐IR 8400CE) [40]. Exactly 1.0 g of extract was placed between the attenuated total reflectance of the instrument equipped with a deuterated triglycine sulphate detector and a potassium bromide beam splitter. The infrared absorption spectrum was obtained at a frequency range between 4000 and 400 cm−1, and the functional groups were determined by comparing the vibration frequency with the IR correlation chart published in the literature [70].

4.4. Phenolic Compounds Identification and Quantification

The identification and quantification of gallic acid, sinapic acid, vanilic acid, ferulic acid, rutin and quercetin in aqueous extracts of Vernonia amygdalina Del., was done using HPLC‐UV‐Vis assay [71, 72]. The instrument (Agress 1100 HPLC; Biobase Biotech Co., Ltd., China) was equipped with model P1100 high‐pressure constant flow pumps and the D1100 UV‐Visible detector. To achieve separation, the extracted diluted sample was filtered using 0.45 µm filter membranes and injected (10.0 µL) into an ODS2 C18 reverse‐phase hypersil column (250 × 4.6 mm, 5 µm) at ambient temperature and a flow rate of 0.9 mL/min. The compounds were identified using a binary mobile phase of solvent A (0.7% acetic acid in water, pH 2.5) and solvent B (100% methanol). The system of solvent gradients was programmed for 30 min as follows: 0.1 min 10.0% B; 5 min 30% B; 8 min 50% B; 13 min 90% B; 20 min 50% B; 25 min 10% B; 30 min 10% B. The compounds were identified by comparing UV spectra and chromatographic retention times recorded at 280 nm, and quantified using standard calibration curves. To optimise and validate the method, the limit of detection (LOD) and limit of quantification (LOQ) were calculated from the signal‐to‐noise (S/N) ratio of 3 for the LOD and 10 for LOQ for each compound: LOD = y + 3SD and LOQ = y + 10SD, where y is the signal of the blank and SD is the standard deviation of the least concentrated analyte. Recovery tests of compounds were carried out by spiking the sample with 1 mL of gallic acid, sinapic acid, vanillic acid, ferulic acid, rutin and quercetin. Recoveries were calculated as: R (%) = (Ca—Cb)/Cc, with Ca as the measured concentration in the sample after spiking, Cb as the measured concentration in the sample before spiking and Cc as the concentration of the reference standard used for spiking.

4.5. Volatile Phytochemicals Identification

To extract volatile compounds for GC‐MS analysis, 1 ppm of the extract was prepared by dissolving the freeze‐dried extract in methanol (99.9%, LC‐MS grade). Extracted volatile phytochemicals were analysed with the GC‐MS system (Agilent 7890 GC) coupled with a 5975C mass selective detector [73]. The instrument used helium as a gas carrier (99.9%) with a flow rate of 1.0 mL/min and an injection volume of 1 µL of the sample in methanol through a Zebron‐5MS capillary column (zb‐5msi 30 m x 0.25 x mm x 0.25 µm). The oven temperature was set to increase from 40°C to 280°C at a rate of 2°C/min. The mass spectrometer was operated with a scan range of 40–400 m/z in electron impact mode at 70 eV for 42 min. The compounds were identified by comparing mass spectra with the NIST and GC–MS libraries of the mass finder [74].

4.6. Data Management and Analysis

One‐way analysis of variance was used to compare the means of HPLC—UV‐Vis results, and post hoc (Tukey's test) was employed to separate the means using GraphPad Prism version 10.1.2 (San Diego, USA). Values of p < 0.05 were considered statistically significant, and the results presented are mean ± standard deviation in triplicate.

Funding

The authors would like to thank Mzuzu University through the African Centre of Excellence in Neglected and Underutilised Biodiversity (ACENUB) for its financial support.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors would like to thank Mzuzu University through the African Centre of Excellence in Neglected and Underutilised Biodiversity (ACENUB) for its financial support. We thank the Department of Basic Sciences at the Lilongwe University of Agriculture and Natural Resources and the Chemistry Department of the University of Malawi for supporting the study through laboratory facilities.

Data Availability Statement

The data have been shown in text. Any other additional data may be made available upon formal request if needed.

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