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Article

Phytochemical Characterization and Antioxidant Activity of Cajanus cajan Leaf Extracts for Nutraceutical Applications

by
Mariel Monrroy
1,2,3,* and
José Renán García
1,2,3,*
1
Research Center in Biochemistry and Applied Chemistry, Faculty of Natural and Exact Sciences, Autonomous University of Chiriqui, David P.O. Box 0427, Panama
2
Department of Chemistry, Faculty of Natural and Exact Sciences, Autonomous University of Chiriqui, David P.O. Box 0427, Panama
3
National Research System (SNI), National Secretariat of Science, Technology and Innovation (SENACYT), Panama City 0816-02852, Panama
*
Authors to whom correspondence should be addressed.
Molecules 2025, 30(8), 1773; https://doi.org/10.3390/molecules30081773
Submission received: 6 March 2025 / Revised: 5 April 2025 / Accepted: 13 April 2025 / Published: 15 April 2025
(This article belongs to the Special Issue Natural Antioxidants in Functional Food)
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Abstract

:
Cajanus cajan (guandú) is a widely cultivated leguminous plant in Panama; however, its phytochemical composition remains underexplored. Traditionally used in Asia and Africa for its medicinal properties, the plant’s bioactive compounds responsible for these benefits have not been fully identified. The phytochemical profile and antioxidant capacity of C. cajan leaf extracts from Panama were characterized, highlighting their potential applications. Ethanolic extracts obtained via ultrasonication were analyzed through phytochemical screening, confirming the presence of alkaloids, tannins, saponins, and steroids. Spectrophotometric analysis revealed high total phenolic (71 mg g−1) and flavonoid (30 mg g−1) contents. Antioxidant assays demonstrated significant 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+) inhibition and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Gas chromatography–mass spectrometry (GC-MS) analysis identified 35 bioactive compounds in C. cajan leaves for the first time, including lupeol (antimicrobial and antitumor), lupenone (antidiabetic), squalene (antitumor and antioxidant), tocopherol (antioxidant), and β-amyrin (antibacterial and anti-Alzheimer’s). These findings expand the known phytochemical profile of C. cajan, supporting its pharmaceutical, nutraceutical, and agro-industrial potential. Moreover, this research provides a foundation for further studies on the plant’s bioactive compounds and their applications in human health and sustainable agriculture.

1. Introduction

Cajanus cajan (L.) Millsp., commonly known as “guandú”, “gandul”, or “pigeonpea”, is a perennial leguminous plant widely cultivated in tropical and subtropical regions [1]. It is the sixth most produced food legume globally, following common beans (Phaseolus vulgaris L.), chickpeas (Cicer arietinum L.), field peas (Pisum sativum L.), cowpeas (Vigna unguiculata), and lentil peas (Lens culinaris) [2]. Cultivation of C. cajan spans approximately 5.4 million hectares worldwide, yielding over 4 million tons annually, with India accounting for more than 79% of global production [3]. Beyond its role in food security, C. cajan is valued for its soil-enriching properties through nitrogen fixation and its high nutritional content, providing essential minerals, B vitamins, and proteins [4].
In addition to its nutritional significance, C. cajan has traditionally been used in Asian and African medicine for treating various diseases, such as diabetes, ulcers, skin irritation, hepatitis, measles, jaundice, dysentery, and nervous disorders [1,5]. Different parts of the plant exhibit medicinal properties with widely documented medicinal applications. The leaves alleviate urinary discomfort, chronic joint pain, diabetes, inflammation, ulcers, malaria, epilepsy, colds, bronchitis, poisoning, and genital and skin inflammation. They also possess laxative, coagulant, analgesic, diuretic, and hypocholesterolemic properties [1,6,7]. The flowers are used to alleviate respiratory infections, colds, pneumonia, bronchitis, and menstrual disorders [8]. The seeds function as energy stimulants, poultices, and anti-dizziness remedies [1,9]. Furthermore, the roots are traditionally used as an expectorant, antiparasitic, and treatment for syphilis and poisoning [8]. Additionally, C. cajan has been reported to aid in treating gingivitis and stomatitis, stimulate lactation, promote oral hygiene, and exhibit antimicrobial, anti-inflammatory, antioxidant, and anticancer properties [10,11].
Building on the therapeutic properties of C. cajan, studies conducted in India, China, and Nigeria have demonstrated the antimicrobial potential of ethanolic and methanolic extracts of C. cajan leaves, seeds, and roots. These extracts have shown antimicrobial activity against various microorganisms, including Staphylococcus aureus, Bacillus subtilis, Streptococcus sp., Salmonella thypi, Klebsiella sp., and Escherichia coli [12,13]. Despite these promising findings, the medicinal properties of C. cajan remain partially understood, as not all effects have been associated with specific molecules.
The plant, especially its leaves, is a rich source of bioactive compounds, including flavonoids, stilbenes, isoflavones, coumarins, and triterpenes, contributing to its pharmacological potential [6,14]. Among these, phenolic compounds and flavonoids play a crucial role in antioxidant activity by scavenging free radicals and reducing oxidative stress, which is associated with various chronic diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions [15,16,17]. These compounds have demonstrated potent antioxidant, anticancer, and antimicrobial properties that can modulate lipid metabolism and inflammatory responses [5,6,18].
Despite these promising findings, the phytochemical composition of C. cajan remains underexplored outside of Asia and Africa. The concentration and profile of its bioactive compounds can vary based on geographic location, soil composition, plant cultivar, and agricultural practices. However, few studies have examined C. cajan in Panama and Latin America. To address this gap, the present study aims to chemically characterize C. cajan leaf extracts from Panama by determining their phenolic, flavonoid, and anthocyanin content, as well as evaluating their antioxidant activity using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. These findings contribute to the understanding of the plant’s phytochemical profile and its potential for future nutraceutical and biotechnological research.

2. Results

2.1. Chemical Composition of C. cajan Leaves

Carbohydrate analysis revealed glucose and mannose in the C. cajan leaves, with concentrations of 134 and 56 g kg−1, respectively. The Fourier transform infrared (FT-IR) spectrum of the C. cajan leaves revealed functional groups characteristic of lignocellulosic materials (Figure 1). The absorption bands at 3289 and 2920 cm−1 correspond to the stretching vibrations of the hydroxyl and aliphatic C–H functional groups, respectively, suggesting the presence of carbohydrates or amino acids, and lipids [19]. The band at 1707 cm−1 is associated with C=O stretching vibrations of the ester carbonyl or carboxylic acid functional group. Additionally, the –C=O absorption can be assigned to conjugated or nonconjugated, saturated or unsaturated amides, acids, or other systems. Peaks associated with aromatic C stretching were identified at 1545 and 1440 cm−1, whereas the peak at 1314 cm−1 can be attributed to C–H asymmetric deformation and COO− anion stretching. The bands in the 1440–1201 cm−1 range were assigned to C–O stretching and O–H deformation vibrations, which are likely part of the flavonoid structure [19]. Finally, the absorption band at 1024 cm−1 corresponds to the C–O stretching of either an ester or an ether, and the band at 752 cm−1 is attributed to CH bending.
Figure 2 presents the phytochemical analysis results of the C. cajan extract, revealing the presence of several bioactive compounds, including alkaloids, flavonoids, tannins, and terpenes/steroids.

2.2. Phenolic Compounds, Flavonoids, and Anthocyanin Content

Ultrasound-assisted extraction using a 60% hydroethanolic solution yielded a total phenolic content (TPC) of 72 ± 1 mg gallic acid equivalents (GAE) g−1, a total flavonoid content (TFC) of 30.4 ± 0.5 mg quercetin equivalents (QE) g−1, and a total anthocyanin content (TAC) of 0.41 ± 0.01 mg g−1, confirming the bioactive potential of C. cajan leaves. A comparison of TPC and TFC values with other medicinal herbs is provided in Figure 3.

2.3. C. cajan Leaves Exhibit High Antioxidant Activity According to ABTS and DPPH Assays

The antioxidant activity of C. cajan leaves was evaluated using ABTS and DPPH assays, which demonstrated that the leaf extract exhibited strong antioxidant activity, with a Trolox equivalent antioxidant capacity (TEAC) value of 11.5 ± 0.2 mM Trolox equivalents (TE) g−1 in the ABTS radical cation (ABTS+) scavenging assay and 289 ± 1 µM TE g−1 in the DPPH assay. Notably, the TEAC values from the ABTS assay were significantly higher (p < 0.05) than those from the DPPH assay. The antioxidant capacity of C. cajan leaves was then compared to that of commonly consumed berries, such as blackberries, cranberries, blueberries, and blackcurrants (Figure 4).

2.4. Identification of Active Compounds by GC-MS

Gas chromatography–mass spectrometry (GC-MS) analysis provides valuable insight into the volatile organic compounds in C. cajan leaves, revealing a diverse array of bioactive molecules with potential pharmacological and industrial applications. The total ion chromatograms (Figure 5) depict the signals corresponding to these active compounds, which were categorized based on their elution time, molecular formula, molecular weight, and fragment ions (Table 1). The bioactive compounds were identified by GC-MS analysis with a similarity index of >85%. This comprehensive characterization lays the foundation for further exploration of individual compounds and their respective biological activities.
Among the 44 compounds identified, several have been previously reported in C. cajan, including hexadecanoic acid, methyl palmitate, ethyl palmitate, α-guaiene, pinostrobin chalcone, 2,4-di-tert-butylphenol, phytol, α-selinene, and α-himachalene [1,11,18,22,23,24,25]. However, this current study expands the list by identifying 35 new compounds in C. cajan leaves, comprising terpenoids (sesquiterpenes, diterpenes, and triterpenes), phenolic compounds, flavonoids, and fatty acids. Notably, several of these compounds have also been reported in various plant species, fungi, and bacteria and have individually been shown to exhibit antioxidant, anti-inflammatory, antimicrobial, and anticancer properties (Table 2).

3. Discussion

The measured carbohydrate content of C. cajan leaves exceeded the total carbohydrate content of 63 g kg−1 reported by Sahu et al. [85] in India but was lower than the 656 g kg−1 reported by Yang et al. [86] in Taiwan. Such variations may be attributed to differences in plant variety, geographical origin, and environmental conditions, which influence carbohydrate content. These findings highlight the significant variability in carbohydrate composition across different regions and cultivation practices, underscoring its potential implications for both the nutritional and medicinal uses of C. cajan.
The FT-IR spectrum of C. cajan also revealed the presence of functional groups, such as polyphenols, pyranose, and fatty acids. Similar spectra have been reported for other plants and lignocellulosic materials, highlighting their structural similarities and the presence of compounds with potential antioxidant and antimicrobial properties [19,87].
The phytochemical composition of C. cajan includes alkaloids, flavonoids, tannins, and terpenes/steroids and is consistent with previous findings by Anadebe et al. [24]. Similarly, Sahu et al. [85] and Devi et al. [12] reported the presence of alkaloids, tannins, flavonoids, and saponins but did not identify terpenes. These variations may be attributed to differences in extraction methods, highlighting the influence of extraction techniques on the composition of bioactive compounds in C. cajan.
The observed TPC and TFC values confirm the bioactive potential of C. cajan leaves and align with or exceed previously reported data. For comparison, Devi et al. [88] reported a TPC of 55 mg GAE g−1 and TFC of 36 mg QE g−1 using maceration extraction with continuous stirring and ethanol as the solvent. Yang et al. [86] reported TPC values of 13.5 and 7.2 mg GAE g−1 using two extraction methods: hot water extraction (decoction) and maceration with 50% ethanol. Also, Yang et al. [86] reported TFC values of 10.4 and 0.2 mg QE g−1 by extraction with ethanol and water, respectively, while Aja et al. [89] reported a TFC of 4.2 mg QE g−1 and TAC of 0.08 mg g−1 by maceration with ethyl acetate (EtOAc). These discrepancies highlight the influence of the extraction techniques on the yield of bioactive compounds. Ultrasonication-assisted extraction, as used in our study, has been reported to enhance cell wall disruption, facilitating the release of phenolic compounds and flavonoids [90], which may explain the higher concentrations observed. However, beyond extraction methods, the concentration and composition of these bioactive compounds are subject to environmental and agronomic factors. Differences in soil composition, climatic conditions, geographic location, and plant cultivar can significantly affect the secondary metabolite profile of C. cajan [91,92,93]. For example, studies have demonstrated that phenolic compound accumulation is affected by UV exposure, temperature fluctuations, and soil nutrient availability [91,92,94], further contributing to the discrepancies observed across different studies.
The TPC and TFC values in C. cajan leaves were notably higher than those reported for medicinal herbs of the Rosaceae, Asteraceae, and Lamiaceae families (Figure 4). For example, Sytar et al. [20] found TPC values ranging from 1 to 18.6 mg GAE g−1 and TFC values between 0.2 and 11.1 mg QE g−1 in leaf extracts of these families. These findings suggest that C. cajan is a rich source of natural antioxidants, reinforcing its pharmacological properties and traditional medicinal uses.
The presence of phenolic compounds, flavonoids, and anthocyanins in C. cajan leaf extract validates its antioxidant and anti-inflammatory potential, positioning it as a promising candidate for nutraceutical and functional food development. Future studies should focus on isolating and characterizing the specific bioactive components responsible for these properties and optimizing extraction techniques to maximize yield and bioactivity.
The antioxidant activity of C. cajan leaves was evaluated using ABTS and DPPH assays, both of which operate through the single-electron transfer (SET) mechanism to assess the ability of a substance to donate electrons, neutralizing free radicals and reactive species. These assays provide valuable insights into the plant’s potential to combat oxidative stress [87,95]. Since ABTS and DPPH assays target different radicals, they offer a more comprehensive assessment of antioxidant capacity. The differences between the ABTS and DPPH values in C. cajan leaf extract may be attributed to variations in the solubility and reactivity of antioxidants in aqueous versus lipophilic systems. The ABTS assay, which operates in an aqueous system, can better represent the activity of hydrophilic antioxidants, while the DPPH assay primarily interacts with lipophilic compounds, providing a complementary measure of antioxidant capacity.
Interestingly, the ABTS antioxidant capacity of C. cajan leaves was significantly higher (p < 0.05) than that of commonly consumed berries, such as blackberries, cranberries, blueberries, and blackcurrants, which are widely recognized for their high antioxidant content and thus serve as excellent references in antioxidant studies. These berries exhibit TEAC values of 464 ± 6, 376 ± 6, 261 ± 6, and 230 ± 5 µM TE g−1, respectively, in the ABTS assay [21]. Despite C. cajan exhibiting lower values (p < 0.05) for the DPPH assay compared to the berries, except blackcurrants, its high antioxidant capacity based on the ABTS assay indicates that it is a highly promising natural source of antioxidants.
The remarkable antioxidant activity of C. cajan can be attributed to its high phenolic and flavonoid contents, which are well known for their ability to neutralize free radicals and prevent oxidative damage [96], which contribute to the development of various chronic diseases, such as cancer, cardiovascular disease, and neurodegenerative disorders [15,16,17,97]. This positions C. cajan as a valuable candidate for the development of functional foods and nutraceuticals. Using C. cajan in preventive health strategies could help mitigate the risk of oxidative stress-related diseases, offering a natural plant-based solution for improving human health and well-being.
Further research on the specific bioactive components responsible for these effects and their bioavailability and stability in various formulations is essential to optimize C. cajan as a therapeutic agent. Additionally, exploring the interaction of these antioxidants with other bioactive compounds present in plants may provide further insights into their synergistic effects and overall health benefits.
The identification of new compounds in C. cajan leaves through GC-MS represents a significant contribution to the understanding of this plant’s bioactive potential. While these compounds have been reported in other plant species, their identification in C. cajan highlights a novel aspect of this study. The biological activities associated with these compounds, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties (Table 2), open new possibilities for the therapeutic application of C. cajan leaf extracts. Notably, compounds like α-selinene, oleamide, and levoglucosenone exhibit promising biological activities such as anti-plasmodial, anti-inflammatory, neuroprotective, and bio-based drug discovery potential [29,30,69,70]. Additionally, the identification of triterpenoids like lupeol and lupenone offers exciting prospects for cardiovascular protection, anti-inflammatory action, and anticancer therapy [53,54,55,56]. The discovery of fatty acids, including methyl palmitate and ethyl linoleate, suggests additional therapeutic benefits, including neuroprotective and cardioprotective activity, as well as insecticidal, antibacterial, and anti-inflammatory properties [63,64,67,68]. Interestingly, certain phthalates, such as dibutyl phthalate, di-isobutyl phthalate, and dioctyl phthalate, were also detected. Despite concerns regarding their potential endocrine-disrupting effects, particularly with di-isobutyl phthalate, these compounds offer valuable industrial applications as plasticizers, biosensors, and in other technologies, underscoring their importance in various fields [75,76,77,78,82,83,84]. Additionally, compounds like 1,3-ditert-butylbenzene and 1-pentadecene suggest the potential use of C. cajan in natural pest control [71,72]. Furthermore, biofuel-related compounds such as heptadecane reinforce the possibility of utilization in renewable energy research [47,48].
These diverse compounds collectively underscore the untapped therapeutic, agricultural, and industrial potential of C. cajan leaf extracts, positioning the plant as a promising candidate for further research. Future studies should focus on isolating, characterizing, and understanding the mechanisms of action of these compounds to optimize their applications in pharmaceuticals, nutraceuticals, and agro-industrial sectors.
While the phytochemical profile of C. cajan has been widely studied, particularly in research conducted in India and Africa, data on this species in the American region, including Panama, remain scarce. Environmental factors such as climate, soil composition, and geographical location significantly influence the production and accumulation of bioactive compounds, making it essential to explore C. cajan under distinct ecological conditions. In this study, we identified new compounds that had not previously been reported in C. cajan, highlighting the potential variability in its chemical composition across geographical regions. These findings contribute to filling the existing knowledge gap and emphasize the need for further investigation into how regional variations may affect the phytochemical diversity and pharmacological properties of C. cajan.

4. Materials and Methods

4.1. Chemicals and Reagents

All the chemicals and reagents used were of analytical grade and were purchased from reputable suppliers. The following chemicals and reagents were used in the experiments: H2SO4, HCl, KCl, CH3COONa, Na2CO3, NaNO2, and ethanol from Ensure; AlCl3, FeCl3, and NaOH from PanReac AppliChem (Darmstadt, Germany); Folin–Ciocalteu reagent, DPPH, and ABTS from Sigma-Aldrich (Saint Louis, MO, USA); and ethyl acetate, hexane, and chloroform from Merck (Boston, MA, USA). Glucose, mannose, arabinose, and xylose were used as external calibration standards, along with gallic acid, quercetin, and Trolox (Sigma-Aldrich).

4.2. Raw Materials

Fresh C. cajan leaves were harvested from the New Mexican Township, Alanje District, Chiriqui Province (8°25′12.2″ N, 82°43′42.7″ W). The region has a tropical climate, with average temperatures ranging from a maximum of 34 °C to a minimum of 23 °C. The leaves were harvested during the rainy season and collected from agricultural land at an elevation of 15 m above sea level. The soil was sandy loam with a pH of 5.4 and an organic matter (MO) content of 6.4%. The leaves were dried at 60 °C for 48 h, then milled and stored in dark plastic bags under dry conditions until use. Sample analysis started immediately after collection.

4.3. Determination of Carbohydrate Composition and Chemical Characterization

The carbohydrate composition was determined following the methodology of Monrroy et al. [98], with modifications. A 300 mg sample was weighed into a test tube, and 3 mL of 72% (w/w) H2SO4 was added. Hydrolysis was performed in a water bath (WD10G11B, Thermo Scientific, Waltham, MA, USA) at 30 °C for 1 h and stirred every 10 min. The acid was then diluted to 3% (w/w) by adding 79 mL of distilled water, and the mixture was transferred to a 250 mL Erlenmeyer flask and autoclaved (25 X-1, All American, Manitowoc, WI, USA) at 121 °C for 40 min. After cooling, the residual material was filtered through a sintered glass filter (No. 4). The concentration of monomeric sugars in the soluble fraction was analyzed using high-performance liquid chromatography (HPLC) (1260 Infinity, Agilent, Santa Clara, CA, USA) with a Hi Plex-H column at 65 °C at a flow rate of 0.4 mL min−1 and 8.5 mmol L−1 H2SO4. A refractive index detector (G7162A, Agilent, USA) was used, and glucose, mannose, arabinose, and xylose served as external calibration standards. The carbohydrate analysis was performed in triplicate.
Infrared spectra of the samples were obtained using attenuated total reflectance (ATR). Spectra were recorded between 4000 and 650 cm−1 using an FT-IR spectrophotometer (Cary 630, Agilent Technologies, USA) equipped with a deuterated triglycine sulfate detector. The diamond ATR sensor was cleaned with ethyl alcohol before each measurement. The spectral resolution was set to 4 cm−1, with 64 scans performed for each spectrum.
The samples were subjected to phytochemical screening by conducting Dragendorff, Wagner, and Mayer tests for alkaloids, the Shinoda test for flavonoids, the ferric chloride test for tannins, and the Salkowski test for sterols, following the methods described by Devi et al. [12] and Khan et al. [99].

4.4. Extraction of Bioactive Compounds Using Ultrasound-Assisted Extraction

The extracts were obtained via ultrasound-assisted extraction from hydroethanolic solutions. Specifically, 0.5 g of the dried sample was sonicated in 10 mL of 600 g L−1 hydroethanolic solution for 20 min in an ultrasonic bath (S60H, Elma, Singen, Baden-Württemberg, Germany). The resulting extracts were then cooled and filtered.

4.5. Phenolic Compounds, Flavonoids, and Anthocyanin Content Evaluation

As described previously, TPC was determined using the Folin–Ciocalteu assay [87]. Different diluted extracts (500 μL) were mixed with 2500 μL of Folin–Ciocalteu reagent (0.2 mol L−1) and allowed to stand for 5 min. Then, 2 mL of 75 g kg−1 sodium carbonate solution was added, and the mixture was incubated in the dark at 25 °C for 2 h. The absorbance of the solutions was measured at 754 nm using a visible light spectrophotometer (Genesys 10S, Thermo Scientific, USA). A calibration curve was prepared using gallic acid solutions (0–12 µg mL−1), and results were expressed as milligrams of GAE per gram of dry peel mass.
TFC was determined spectrophotometrically using the AlCl3 colorimetric method [100], with modifications. Briefly, 50 µL of the extract was diluted to 5 mL with distilled water, followed by adding 0.3 mL of 50 g L−1 NaNO2. After 5 min, 0.3 mL of 100 g L−1 of AlCl3 was added to the mixture. Shortly thereafter, 1 mol L−1 sodium hydroxide NaOH was added, and distilled water was added to bring the total solution volume to 10 mL. After 15 min, the absorbance of the solution was measured at 374 nm using an ultraviolet spectrophotometer (Genesys 10S; Thermo Scientific, USA). A calibration curve was constructed using quercetin solutions (0–15 µg mL−1), and the flavonoid content was expressed as milligrams of QE per gram of dry mass.
TAC was estimated using the pH differential method [87]. Extract aliquots were adjusted to pH 1.0 and 4.5 using 25 mM KCl and 0.4 M CH3COONa buffer solutions, respectively, and allowed to equilibrate for 20 min. Absorbance was measured at 520 and 700 nm. TAC was calculated based on the difference in absorbances at pH 1.0 and 4.5, using the molecular weight of cyanidin-3-glucoside (449.2 g mol−1), the dilution factor (DF), and the molar extinction coefficient of cyanidin-3-glucoside (26,900 L mol−1 cm−1).
Each analysis was performed in sextuplicate to ensure the reliability and reproducibility of the results.

4.6. Determination of Antioxidant Activity Using the ABTS and DPPH Assays

The ABTS assay was performed using the method described by Re et al. [101]. The ABTS radical cation was generated by mixing equal portions of 7 mM ABTS solution and 2.45 mM potassium persulfate solution. The mixture was incubated in the dark at 25 °C for 16 h and diluted with 96% ethanol until its absorbance at 754 nm reached 0.7 ± 0.02. For the assay, 1900 µL of the ABTS•+ solution was mixed with 100 µL of the diluted extract and incubated at room temperature in the dark for 10 min. The absorbance of the samples was measured at 754 nm using a visible light spectrophotometer (Genesys 10S, Thermo Scientific, USA). ABTS•+ scavenging activity was calculated by comparison with a standard curve of Trolox (40 and 320 mmol L−1 Trolox), and the results were expressed as mmol of TE per gram of dry peel mass (mmol TE g−1).
The DPPH assay was conducted according to Monrroy et al. [87] and Thaipong et al. [102]. A DPPH radical solution was prepared by dissolving 13 mg DPPH in 100 mL methanol. To achieve an absorbance of 0.70 ± 0.02 at 515 nm, 10 mL of this solution was diluted with 45 mL of methanol. For the assay, 100 µL of the extract was mixed with 2900 µL of the DPPH solution and incubated in the dark at room temperature for 30 min. The absorbance of the sample was measured at 515 nm. DPPH scavenging activity was calculated by comparison with a standard curve of Trolox (40 and 600 mmol L−1 Trolox). The results are expressed as mmol of TE per gram of dry peel mass (mmol TE g−1).
Both assays were performed in sextuplicate.

4.7. Isolation and Purification of Bioactive Compounds via Liquid–Liquid Extraction (LLE)

The hydroethanolic extract was fractioned using EtOAc and hexane. Initially, the extract was evaporated, and the resulting residue was resuspended in water. The aqueous extract (3 mL) was acidified to a pH of 2 with HCl and subjected to liquid–liquid extraction (LLE) with EtOAc (3 × 3 mL). The EtOAc phase was treated with activated charcoal to absorb the pigments, filtered, dried over anhydrous Na2SO4, and evaporated. The remaining residue was resuspended in ethanol for GC-MS analysis. The LLE process was then repeated using hexane.

4.8. Identification of Bioactive Compounds Using GC-MS

The bioactive compounds obtained through LLE with EtOAc and hexane were analyzed using a 7890A GC system coupled with 5975C MS (Agilent Technologies, USA) fitted with an HP-5MS column (30 m in length × 250 μm in diameter × 0.25 μm film thickness). Pure He was used as the carrier gas at a constant flow rate of 1 mL min−1. Furthermore, 1 μL of the extract was injected in splitless mode, with ionization of the sample components performed at 70 eV. The oven temperature was initially maintained at 60 °C, then increased at 10 °C min−1 to a final temperature of 280 °C over 30 min. Mass spectra were recorded in the m/z range of 45–550 at a scan rate of two scans per second. The total GC runtime was 52 min. Compound identification was performed by comparing the mass spectra with those in the NIST05 Mass Spectral Library.

4.9. Statistical Analysis of Antioxidant Activity and Literature Comparison

T-tests were used to determine differences in antioxidant activity (measured by ABTS and DPPH assays) and to compare with the data reported in the literature. The results were considered statistically significant at p < 0.05. Statistical analyses were performed using Statgraphics Centurion XVIII software, version 18.1.16 (Statgraphics Technologies, The Plains, VA, USA).

5. Conclusions

An in-depth characterization of the active compounds in Panamanian C. cajan leaves was conducted, exhibiting their potential for various applications. Using ethanolic extraction assisted by ultrasonication, phytochemical analysis, and visible spectrophotometry, significant secondary metabolites such as alkaloids, tannins, saponins, and steroids were identified. Remarkably, high levels of phenolic and flavonoid contents were observed, contributing to the exceptional antioxidant capacity of the leaf extract.
Furthermore, GC-MS analysis revealed 31 bioactive compounds, many of which have never been reported in C. cajan leaves before. These identified compounds demonstrated multiple promising biological activities, including antimicrobial, antitumor, anti-inflammatory, and antioxidant effects. These properties underscore C. cajan’s potential as a source of bioactive molecules for developing novel medicinal and therapeutic products.
The unique environmental conditions of Panama, including its climate, soil composition, and geographical location, may have contributed to the distinct phytochemical profile observed in this study. These geographic and ecological factors can influence the production of bioactive compounds, further emphasizing the importance of investigating C. cajan across different regions. These findings not only expand the knowledge of the nutraceutical and ethnobotanical properties of C. cajan but also identify potential biotechnological applications that could strengthen the agricultural sector in Panama.
Therefore, a solid foundation is established for future research into the pharmacological, agricultural, and industrial potential of C. cajan, encouraging its broader use in health applications and agronomy. Further investigation into the bioactive compounds of C. cajan, particularly in relation to the environmental factors of different geographical regions, will be essential for fully harnessing its potential across various industries, positioning the plant as a vital resource for sustainable innovation.

Author Contributions

Conceptualization, M.M. and J.R.G.; methodology, M.M. and J.R.G.; formal analysis, M.M. and J.R.G.; investigation, M.M. and J.R.G.; writing—original draft preparation, M.M. and J.R.G.; writing—review and editing, M.M.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Secretariat of Science, Technology, and Innovation (SENACYT) as members of the Panama Research National System (SNI) and Grant PFID-FID-2021-185, and Vice rectorate for research and postgraduate studies of the Autonomous University of Chiriqui (UNACHI), for Grant 184-CN-03-C202-05-2016.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors thank analysts Rosa Parada and Glenda Maldonado, as well as Juanita Freer of the Renewable Resources Laboratory, Biotechnology Center, Universidad de Concepcion-Chile, which is responsible for supporting the identification of compounds using GC-MS. Additionally, we thank the Department of Chemistry at UNACHI.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ABTS2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
ABTS+ABTS radical cation
DPPH2,2-diphenyl-1-picrylhydrazyl
GC-MS Gas chromatography–mass spectrometry
FT-IRFourier transform infrared
TPCTotal phenolic content
GAEGallic acid equivalents
TFCTotal flavonoid content
QEQuercetin equivalents
TACTotal anthocyanin content
TEACTrolox equivalent antioxidant capacity
TETrolox equivalents
LLELiquid–liquid extraction
EtOAcEthyl acetate
SETSingle-electron transfer
HPLCHigh-performance liquid chromatography
ATRAttenuated total reflectance

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Molecules 30 01773 g001
Figure 1. FT-IR spectrum of Cajanus cajan (L.) Millsp.
Figure 1. FT-IR spectrum of Cajanus cajan (L.) Millsp.
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Figure 2. The phytochemical analysis of C. cajan leaves shows the presence (red) and absence (green) of phytochemicals in six qualitative tests.
Figure 2. The phytochemical analysis of C. cajan leaves shows the presence (red) and absence (green) of phytochemicals in six qualitative tests.
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Molecules 30 01773 g003
Figure 3. Total phenolic content (TPC) and total flavonoid content (TFC) of C. cajan leaves compared to those of other medicinal herbs. The values for medicinal herbs were obtained from Sytar et al. [20]. Values are given as mean ± standard deviation.
Figure 3. Total phenolic content (TPC) and total flavonoid content (TFC) of C. cajan leaves compared to those of other medicinal herbs. The values for medicinal herbs were obtained from Sytar et al. [20]. Values are given as mean ± standard deviation.
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Molecules 30 01773 g004
Figure 4. Antioxidant capacity of C. cajan leaves compared to common berries, known for their high antioxidant content. Berry values were obtained from Kim et al. [21]. Values are given as mean ± standard deviation (n = 6 for C. cajan leaves and n = 5 for berries). Significant differences between C. cajan leaves and berries for each assay (ABTS and DPPH) are indicated by different letters above the bars for each assay (p < 0.05).
Figure 4. Antioxidant capacity of C. cajan leaves compared to common berries, known for their high antioxidant content. Berry values were obtained from Kim et al. [21]. Values are given as mean ± standard deviation (n = 6 for C. cajan leaves and n = 5 for berries). Significant differences between C. cajan leaves and berries for each assay (ABTS and DPPH) are indicated by different letters above the bars for each assay (p < 0.05).
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Molecules 30 01773 g005
Figure 5. Total ion chromatograms obtained by gas chromatography–mass spectrometry (GC-MS) of C. cajan extracts. (A) Hydroethanolic crude extract, (B) ethanolic extract obtained by liquid–liquid extraction (LLE) with ethyl acetate (EtOAc), and (C) ethanolic extract obtained by LLE with hexane. The chromatograms display molecular ion peaks corresponding to the various compounds present in each extract. Differences in the profiles reflect variations in the chemical composition of the extracts based on the extraction solvent used. This analysis highlights the diversity of bioactive compounds in C. cajan leaves.
Figure 5. Total ion chromatograms obtained by gas chromatography–mass spectrometry (GC-MS) of C. cajan extracts. (A) Hydroethanolic crude extract, (B) ethanolic extract obtained by liquid–liquid extraction (LLE) with ethyl acetate (EtOAc), and (C) ethanolic extract obtained by LLE with hexane. The chromatograms display molecular ion peaks corresponding to the various compounds present in each extract. Differences in the profiles reflect variations in the chemical composition of the extracts based on the extraction solvent used. This analysis highlights the diversity of bioactive compounds in C. cajan leaves.
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Table 1. Active compounds identified in the mass spectra of C. cajan leaf extracts.
Table 1. Active compounds identified in the mass spectra of C. cajan leaf extracts.
CompoundsCompounds TypeMol. FormulaTr (Min)Peak Area
(%)		Mol. Wt.
(g mol−1)		Top PeakFrag. Ions (m/z)
2nd and 3rd Highest		Extract
α-GuaieneSesquiterpenesC15H2410.410.2204105147 and 107Crude
(−)-α-HimachaleneSesquiterpenesC15H2410.610.2204189119 and 105Crude
cis-(−)-2,4a,5,6,9a-Hexahydro-3,5,5,9-tetramethyl(1H) benzocyclohepteneAromatic hydrocarbonC15H2410.960.120493133 and 105Crude
α -SelineneSesquiterpenesC15H2411.170.120418993 and 204Crude
Phytol Acyclic diterpene alcoholC20H40O15.270.92968281 and 95Crude
Hexadecanoic acidPhenolic acid, saturated fatty acidC16H32O216.091.4 and 4.12567360 and 55Crude and LLE with hexane
Ethyl palmitateFatty acid ethyl esterC18H36O216.370.2, 13.3 and 5.928588101 and 55Crude and LLE with EtOAc and hexane
(E,7R,11R)-Phytol DiterpenoidC20H40O17.530.529671123 and 57Crude and LLE with EtOAc
Linolenyl alcoholFatty primary alcoholC18H30O217.800.52787955 and 95Crude
Ethyl linolenateFatty acid ethyl esterC20H34O218.020.2 and 8.13067967 and 95Crude and LLE with EtOAc
Ethyl stearateFatty acid ethyl esterC20H40O218.220.1, 6.8 and 5.231288101 and 55Crude and LLE with EtOAc and hexane
1-Piperidineacetonitrile,. alpha. -styrylHeterocyclicC15H18N219.399.2226226225 and 165Crude
Pinostrobin chalconeChalconesC16H14O420.1910.9270270193 and 166Crude
4-(4-Methoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolineTetrahydroisoquinolinesC29H50O220.900.829998239 and 57Crude
SqualeneTriterpenoidC30H5023.371.4, 2.8 and 1.044106981 and 95Crude and LLE with EtOAc and hexane
γ-Tocopherol/Vitamin ETocopherolC28H48O226.000.8 and 2.0416416151 and 417Crude and LLE with hexane
n-HeptadecaneAlkaneC17H3626.440.22405771 and 85Crude
Vitamin ETocopherolC29H50O227.221.8431430165 and 432Crude
β-AmyrinPentacyclic triterpenoidC30H50O31.003.5 and 0.7426218203 and 219Crude and LLE with EtOAc
Lup-20(29)-en-3-oneTriterpenoidC30H48O32.1612.7 and 21.0425205109 and 424Crude and LLE with EtOAc
LupeolPentacyclic triterpenoidC30H50O32.773.4 and 6.6426189218 and 207Crude and LLE with EtOAc
2,4-Di-tert-butylphenolPhenolC14H22O11.241.3 and 2.9 206191206 and 57LLE with EtOAc and hexane
Di-isobutyl phthalatePhthalate esterC16H22O415.200.4278149223 and 57LLE with EtOAc
Methyl palmitateFatty acid methyl esterC17H34O215.731.8 and 1.62707487 and 55LLE with EtOAc and hexane
Ethyl 9-hexadecenoateFatty acid esterC18H34O216.301.02825588 and 96LLE with EtOAc
8,11-Octadecadienoic acid, methyl esterFatty acid methyl esterC19H34O217.330.82946781 and 55LLE with EtOAc
cis-11,14,17-Eicosatrienoic acid methyl ester Fatty acid methyl esterC21H36O217.401.03207967and 95LLE with EtOAc
Methyl isostearateEsterC19H38O217.600.7 and 0.52987487 and 298LLE with EtOAc and hexane
Ethyl linoleateFatty acid ethyl esterC20H36O217.934.4 and 1.93086781 and 95LLE with EtOAc and hexane
OleamideFatty amideC18H35NO19.727.8 and 132185972 and 55LLE with EtOAc and hexane
9,12-Octadecadienoic acid, ethyl esterFatty acid ethyl estersC19H38O420.852.933098239 ad 57LLE with EtOAc
Monoethylhexyl phthalic acidMono(2-ethylhexyl) ester of benzene-1,2-dicarboxylic acidC16H22O421.102.4278149167 and 57LLE with EtOAc
β-amyronePentacyclic triterpenesC30H48O31.054.2424218203 and 219LLE with EtOAc
LevoglucosenoneAnhydrohexose and a deoxyketohexoseC6H6O36.151.41269896 and 68LLE with hexane
Benzene, 1,3-bis(1,1-dimethylethyl)-PhenylpropanesC14H227.900.219017557 and 90LLE with hexane
1-PentadeceneAlkeneC15H309.680.42105583 and 69LLE with hexane
PyrogallolPhenolicC6H6O39.916.412612652 and 80LLE with hexane
1-NonadeceneAlkeneC19H3812.100.32668355 and 97LLE with hexane
Di-isobutyl phthalatePhthalate esterC16H22O415.201.127814957 and 150LLE with hexane
Dibutyl phthalatePhthalate esterC16H22O416.152.8278149150 and 205LLE with hexane
8-Octadecenoic acid, methyl esterOleic acid methyl esterC19H36O217.380.62965574 and 69LLE with hexane
Elaidic acidTrans-isomer of oleic acidC18H34O217.772.42825569 and 83LLE with hexane
Ethyl oleateFatty acid ethyl esterC20H38O218.004.93105569 and 83LLE with hexane
Dioctyl phthalatePhthalate esterC24H38O421.151.4390149167 and 279LLE with hexane
Note: The abbreviations used in the table are as follows: Mol. formula (molecular formula of the compound), Tr (min) (retention time in minutes), Mol. wt. (molecular weight), Top peak (main peak identified in the mass spectrum), and Frag. ions (m/z) (fragment ions, indicated as mass-to-charge ratio in the mass spectrum).
Table 2. Active compounds identified in C. cajan leaf extracts and their reported properties and applications.
Table 2. Active compounds identified in C. cajan leaf extracts and their reported properties and applications.
CompoundProperties/UseReferences
α-GuaienePrecursor to rotundone (peppery aroma and flavor). Possesses antimicrobial activity.[26,27]
(−)-α-HimachaleneAntimicrobial agent[28]
α-selineneAnti-plasmodial and anti-inflammatory biomarkers.[29,30]
PhytolPrecursor of vitamin E and vitamin K1. Modulates transcription in cells via transcription factors PPAR-alpha and retinoid X receptor (RXR).
Flavoring agent.
Antimicrobial, cytotoxic, antitumor, antimutagenic, anti-teratogenic, antibiotic-chemotherapeutic, antidiabetic, lipid lowering, antispasmodic, anticonvulsant, antinociceptive, antioxidant, anti-inflammatory, anxiolytic, antidepressant, immunoadjuvancy, hair growth facilitator, hair fall defense and antidandruff activities. Inhibits osteoclast differentiation.		[31,32,33,34]
Hexadecanoic acidAnti-inflammatory agent[35]
Ethyl palmitateAntiviral agent[36]
Linolenyl alcoholAntibacterial and anticancer agent[37,38]
Ethyl linolenateAntinociceptive activity[39]
β-amyroneAnti-inflammatory and anti-obesity agent[40,41]
Ethyl stearateProtective effect against the neurotoxin 6-hydroxydopamine[42]
SqualeneAntitumor, immunity enhancement, antioxidant, detoxifier, skin senility resistance, hypolipidemic, and antibacterial activities.[43]
γ-tocopherolAntioxidant, anti-inflammatory, cancer prevention, and contributions to natriuresis.[44,45,46]
Heptadecane Potential biofuel precursor[47,48]
D-α-TocopherolAntioxidant and modulates lipid peroxidation.[44,49,50]
β-AmyrinAntimicrobial activity and protects against Alzheimer’s disease.[51,52]
LupenoneInnovative drug for preventing and treating diabetic nephropathy.
Anti-inflammatory agent		[53,54]
LupeolAnti-inflammatory, antiapoptotic, anticancer, antioxidant, and antimicrobial activities.
Protective effects on cardiovascular diseases.		[55,56]
2,4-Di-tert-butylphenolAntitubercular, anticancer, antioxidant, anti-inflammatory, insecticidal, herbicidal, and antimicrobial activity.
Exhibits broad toxicity in human and animal cells.		[57,58,59,60]
Di-isobutyl phthalateHuman health risks, including fetal toxicity.[61,62]
Methyl palmitateNeuroprotective and cardioprotective activity.[63,64]
cis-11,14,17-Eicosatrienoic acid methyl esterAntifouling activity.[65]
Methyl isostearateSkin conditioning agent
Emulsifier		[66]
Ethyl LinoleateInsecticidal, antibacterial, and anti-inflammatory activity.[67,68]
OleamideNeuroprotective effects.[69]
LevoglucosenoneBio-based platform for drug discovery.[70]
1,3-ditert-butylbenzeneMosquitocidal activity.[71]
1-PentadeceneInsect repellent.[72]
PyrogallolAntibacterial and antifungal agent.[73,74]
1-NonadeceneAnti-inflammatory activity[60]
Di-isobutyl phthalatePlasticizer and anti-androgenic effects.[75]
Dibutyl phthalateBiosensors, plasticizers, and endocrine disruptor.[76,77,78]
Elaidic acidBoosts tumoral antigen presentation and cancer immunity.
Treatment of colorectal cancer.		[79,80]
Ethyl oleatePromotes the drying of fruits, vegetables, and grains
Effective microemulsion ingredient to increase the bioavailability of a drug
Gasoline additive		[81]
Dioctyl phthalatePlasticizer[82,83,84]
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Monrroy, M.; García, J.R. Phytochemical Characterization and Antioxidant Activity of Cajanus cajan Leaf Extracts for Nutraceutical Applications. Molecules 2025, 30, 1773. https://doi.org/10.3390/molecules30081773

AMA Style

Monrroy M, García JR. Phytochemical Characterization and Antioxidant Activity of Cajanus cajan Leaf Extracts for Nutraceutical Applications. Molecules. 2025; 30(8):1773. https://doi.org/10.3390/molecules30081773

Chicago/Turabian Style

Monrroy, Mariel, and José Renán García. 2025. "Phytochemical Characterization and Antioxidant Activity of Cajanus cajan Leaf Extracts for Nutraceutical Applications" Molecules 30, no. 8: 1773. https://doi.org/10.3390/molecules30081773

APA Style

Monrroy, M., & García, J. R. (2025). Phytochemical Characterization and Antioxidant Activity of Cajanus cajan Leaf Extracts for Nutraceutical Applications. Molecules, 30(8), 1773. https://doi.org/10.3390/molecules30081773

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