Phytochemical and pharmacological aspects of the genus Chamaecrista: a systematic review

3.1.1. Phenolic compounds

Phenolic compounds are widely distributed in the plant kingdom and have garnered significant scientific interest in recent years because of their antioxidant capacity, which neutralizes free radicals in the body [20,21]. This phenomenon has significant health implications, as it is associated with a reduced risk of heart disease, cancer, and other disorders linked to oxidative stress [22,23]. In addition to their antioxidant activity, phenolic compounds exhibit various beneficial properties, including anti-inflammatory, anti-cancer, antimicrobial, and antiviral activities [22,23].

Among the identified phenolic compounds, notable groups included flavones, phenolic acids, flavonols, flavanones, flavanonol derivatives, coumarins, and isocoumarins.

Quirós-Guerrero et al. [24] reported the highest number of identified phenolic compounds in Chamaecrista, using liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) in negative ionization mode from a methanolic extract of aerial parts. They identified a total of 44 metabolites in the species C. nictitans, of which 70% were reported for the first time for this species, as shown in Figure 5 as compounds 6, 7, 10, 12, 14, 16, 17, 18, 21, 22, 25, 27-32, 41, 44–46, 48–50, 54, 55, 65, 67–69.

Figure 5. Phenolic compounds reported in species of Chamaecrista. [Click here to view]

Phenolic compounds have been identified in the ethanolic extract and ethyl acetate fraction of C. diphylla leaves using ultra-high-resolution liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry [25]. Among these, sinapic acid (38) was identified, which shows antibacterial, antihyperglycemic, hepatoprotective, anti-inflammatory, and potentially anticancer properties [26]. Resveratrol (70) is known for its antioxidant and anti-inflammatory effects and ability to protect the cardiovascular system. It has been the subject of numerous studies [27–29]. These findings underscore the richness of phenolic compounds present in C. nictitans. However, the identification of these compounds has been reported in only two species. It is crucial to emphasize the importance of your continued research on other species of the genus Chamaecrista, as it is integral to advancing our understanding of these beneficial compounds.

Flavonoids constitute a vast chemical group of various plant species and are classified into flavones, flavonols, flavanones, isoflavonoids, anthocyanidins, and catechins. They exhibit various pharmacological properties, including antioxidant, diuretic, antispasmodic, antiulcer, and anti-inflammatory [30]. In the studies analyzed, 35% of the chemical groups identified in Chamaecrista species corresponded to flavonoids. For instance, a study on C. duckeana identified 17 flavonoids in methanolic extracts from the stems, roots, fruits, and leaves using UPLC-ESI-HRMS [31]. Most (47%) of these flavonoids were found in the stem extracts (compounds 80, 82, 84, 87, 90, 92, 104, and 113, Table S1), detailed in Figure 5. In comparison, the remaining 47% were distributed among the leaves (compounds 100, 114, and 118, Table S1) and fruit extracts (compounds 84, 85, 111, 115, and 117, Table S1). Another species, C. hildebrandtii, presented 13 compounds, including flavonoids and phenolic derivatives (72, 74, 76, 83, 88, 91, 96, 98, 101, 103, 107–110, Table S1) identified via LC-QTOF-MS in methanolic leaf extracts [32].

Certain compounds have also been identified in several Chamaecrista species. For example, vitexin (119), reported in extracts from C. rotundifolia, C. desvauxii, and C. diphylla, exhibits antimicrobial, anti-inflammatory, and antioxidant effects and is particularly relevant in cosmetology for potential anti-aging benefits [33,34]. In addition, two flavonoids, apigenin, and luteolin, are present in both C. diphylla and C. nictitans extracts [25,35]. Apigenin (79) has antioxidant, anti-inflammatory, and anti-cancer effects, particularly by inhibiting the proliferation of cancer cells in the ovaries, prostate, and colon [36–38]. Luteolin (97) demonstrates various effects, including antioxidant, anti-inflammatory, anti-cardiovascular, anti-cancer, and anti-neurodegenerative properties [38–40]. Isovitexin (94) has been identified in several species, such as C. desvauxii, C. diphylla, C. nictitans, and C. rotundifolia, displays anti-inflammatory and antioxidant effects, and is particularly beneficial in skincare and anti-aging treatments. Among the flavonoids identified in extracts of C. desvauxii, C. nictitans, and C. rotundifolia, quercetin (112) has been widely studied for its antioxidant, anti-inflammatory, and potential anticancer properties [41–43].

Chemical analysis of the Chamaecrista genus has revealed a richness of flavonoids, suggesting its potential application in various fields such as food, cosmetics, and pharmaceutical development. Despite promising research, information remains limited to this genus, and this review represents an initial step toward studying other species within the genus based on reported findings.

3.1.2. Sterols

Sterols are a group of lipids that play crucial roles in the structure of cell membranes and the regulation of various biological processes. They can originate from multiple sources, including animals, plants, and fungi. Those derived from plants are known as phytosterols, which are significant in cholesterol absorption and hormone synthesis. In addition, phytosterols have been observed to exhibit anti-inflammatory, antitumor, and antimicrobial effects, highlighting their broad therapeutic potential in various health areas [44,45].

They were primarily identified in the seed oil of C. absus, representing 5% of the total chemical groups identified in this genus (Table S1), as shown in Figure 6. Notably, three major phytosterols were present: campesterol (124), stigmasterol (127), and β-sitosterol (121), with β-sitosterol being the most abundant. The analysis was performed using gas chromatography with flame ionization detection (GC-FID) [46].

Figure 6. Sterols reported in genus Chamaecrista. [Click here to view]

β-sitosterol is utilized as a nutritional supplement and is recognized for its therapeutic potential, including antioxidant, analgesic, antimicrobial, antidiabetic, and hepatoprotective effects, underscoring its multiple health benefits [47,48].

3.1.3. Fatty acids and aliphatic esters

Fatty acids are essential for human and animal diets and play crucial roles in various physiological functions, such as energy production and cell membrane formation. They are classified as saturated, unsaturated, and polyunsaturated, and balanced consumption promotes optimal cardiovascular health [49]. On the other hand, aliphatic esters are compounds of significant industrial and commercial importance, used as solvents and lubricants in food products, cosmetics, and fragrances. Their versatile properties make them highly valuable in the industry.

Two species, C. absus and C. nigricans, have reported the presence of two fatty acids: n-hexadecanoic acid (compound 133, Table S1) and octadecanoic acid (compound 139, Table S1). In addition, three essential fatty acids were identified in C. absus: α-linolenic acid (Ω 3), linoleic acid (Ω 6), and oleic acid (Ω 9), as shown in Figure 7 [46,50].

Figure 7. Fatty acids and aliphatic esters reported in genus Chamaecrista. [Click here to view]

Alpha-linolenic acid is known for its beneficial properties in preventing and treating cardiovascular and neurodegenerative diseases [51]. It also shows potential anti-inflammatory effects, with preliminary trials reporting its supplementation in patients with SARS-CoV-2 infection to combat inflammation [52]. Moreover, it has been included in the feed of both ruminant and non-ruminant animals, along with linoleic and oleic acids, resulting in improved meat quality in rabbits and poultry, increased egg mass in birds, and enhanced milk quality [53].

Furthermore, the percentage of α-linolenic acid reported in the oil was similar to that found in soybean oil (7%) and slightly lower than that in canola oil (9%), suggesting that it could be a viable alternative [54]. For these reasons, further research on Chamaecrista species is of interest.

3.1.4. Anthraquinones

Anthraquinones are a group of compounds that are widely distributed in nature and found in the roots, leaves, and flowers. These compounds are classified as quinones, benzoquinones, and naphthoquinones. They are known for their pharmacological properties, including anti-inflammatory, antitumor, antimicrobial, and laxative effects [55,56].

Anthraquinones have been reported in three species: C. duckeana, C. nigricans, and C. diphylla, as determined by Ultra-High-Performance liquid chromatography (UPLC-ESI-HRMS), Electron Ionization Mass Spectrometry-m/z, Infrared spectroscopy, and 13C and 1H nuclear magnetic resonance spectrocopy techniques, detailed in Figure 8, respectively (Table S1) [25,31,50].

Figure 8. Anthraquinones reported in genus Chamaecrista. [Click here to view]

Emodin (compound 143, Table S1) has been detected in the leaves of C. diphylla and C. nigricans. Its pharmacological properties include anti-inflammatory, antimutagenic, antimicrobial, antidiabetic, and neuroprotective properties, which help prevent diseases such as Alzheimer’s and Parkinson’s [57,58].

3.1.5. Other chemical compounds

Finally, it is worth mentioning that compounds within the following groups have been reported: triterpene alcohols, xanthone derivatives, triterpene derivatives, and sesquiterpenes, which are compiled in Table S1.

Compounds 146, 149, 150, 152–154, and 157–159 were found in the leaves of C. diphylla, and compounds 147, 155, and 156 were found in the fruits of C. duckeana. The latter was also identified in the stem using UPLC-ESI-HRMS [25,31].

In contrast, compounds 145 and 151 have been reported in the seeds of C. absus using GC-FID and in the leaves of C. nigricans (compound 148) using Gas Chromatography-Mass Spectrometry [46,50].

Among the compounds identified in the genus is β-amyrin, compound 145 ( Fig. 9) reported in C. absus, which decreases blood glucose levels in mice and increases insulin levels [59].

Figure 9. Other chemical compounds reported in different species of Chamaecrista. [Click here to view]

Another compound reported was eupalinolide A (156) in the stems and fruits of C. duckeana. This compound has been studied for its potential anti-inflammatory and antitumor effects [60].

Azelaic acid was identified in the leaf extracts of C. diphylla. It has anti-inflammatory, antioxidant, and antibacterial properties and acne-inhibiting effects and is used to develop cosmetic products [61].

In conclusion, it is essential to highlight the significance of studies on chemical groups reported for the genus Chamaecrista. This genus has gained relevance since a previous review noted some species, emphasizing the importance of Chamaecrista [62]. Even so, it is necessary to continue with future research because 40% of the identified compounds do not report studies on their pharmacological properties, and other chemical groups, such as alkaloids, which have been reported in species belonging to the same subfamily Caesalpinoideae, have not been studied in Chamaecrista. These may represent promising research fields, expanding the knowledge of the pharmacological properties that they may present and their potential applications in health.

In conclusion, advancing the identification and isolation of chemical compounds in Chamaecrista species is essential, especially considering that only one of the studies reviewed has undertaken a comprehensive chemical analysis [24]. The remaining works are largely limited to preliminary identification efforts.

Nevertheless, the field offers significant opportunities for future research. Notably, around 40% of the compounds identified so far have not been investigated for their pharmacological activities. Furthermore, chemical groups such as alkaloids, well-documented in other species within the Caesalpinoideae subfamily, have yet to be explored in Chamaecrista. Addressing these gaps could unlock valuable pharmacological insights and open new pathways for applications in health and medicine. We encourage further multidisciplinary collaborations to fully realize the potential of Chamaecrista species in drug discovery and development [63,64].

3.2.1. Antimicrobial activity

The increasing resistance to conventional antimicrobial agents, mainly owing to their overuse in medicine and agriculture, poses a significant public health challenge, contributing to high morbidity and mortality rates worldwide. This issue also leads to increased healthcare costs and frequent therapeutic failures. Moreover, antimicrobial therapies themselves can cause severe adverse effects, and the use of specific agents is restricted owing to their toxicity [65].

Oil extracted from C. absus seeds exhibited antibacterial activity against a range of gram-positive bacteria, including Staphylococcus aureus, Listeria ivanovii, Listeria innocua, Bacillus subtilis, and Bacillus cereus, as well as the gram-negative bacteria Escherichia coli. The most vigorous activity was observed against L. ivanovii and B. subtilis, with an inhibition zone reaching up to 12 mm, possibly owing to sterols and triterpenoid alcohols in the seed oil [46]. The aqueous extract of C. nigricans leaves, when combined with iron oxide nanoparticles, demonstrated enhanced antibacterial efficacy, producing inhibition of 24 mm against E. coli, 19.5 mm Pseudomonas aeruginosa and 18.5 mm Klebsiella pneumoniae at a concentration of 100 μg/ ml [66]. Similarly, the leaf extract of C. cytisoides displayed notable antibacterial activity, particularly against Micrococcus luteus (21 mm), and inhibited the growth of B. subtilis and E. coli. The comparatively lower activity of many extracts against gram-negative bacteria may be explained by their outer membrane, which acts as an additional barrier to antimicrobial agents [34]. At a concentration of 4 mg/ml, the fruit extract of C. desvauxii inhibited Staphylococcus epidermidis biofilm formation (12.6%). Staphylococcus epidermidis is closely associated with nosocomial infections, and inhibiting virulence factors such as biofilm formation presents an innovative therapeutic approach [33].

Regarding antifungal activity, extracts of C. desvauxii showed inhibition against Candida albicans, with fruit extract being the most effective (19 mm) [34]. These results suggest that different plant parts may exhibit distinct antimicrobial profiles, likely owing to variations in their chemical composition. The methanolic leaf extracts of C. desvauxii, C. nictitans, and Chamaecrista vestita demonstrated both fungistatic and fungicidal activity against dermatophytes such as Trichophyton rubrum, Epidermophyton floccosum, and Trichophyton mentagrophytes at 500 mg/ml, indicating strong anti-dermatophyte potential [34].

The antimicrobial efficacy of Chamaecrista extracts appears to be influenced by the presence and degree of hydroxylation of the phenolic compounds. Nevertheless, there is a notable gap in studies on antifungal activity within this genus, highlighting the need for further research. Natural products from Chamaecrista offer considerable promise owing to their chemical diversity, particularly phenolics with known antifungal properties. Future studies should focus on the isolation and characterization of bioactive compounds, elucidating their mechanisms of action, evaluation using in vivo models, structure-activity relationship studies, and exploration of chemical modifications. Investigating the potential synergistic effects between plant extracts or conventional antimicrobial agents may also reveal new approaches to overcoming microbial resistance.

3.2.2. Anticholinesterase activity

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and the primary cause of dementia worldwide. Current treatments are primarily symptomatic, with acetylcholinesterase (AChE) inhibitors being the most widely prescribed. These agents inhibit AChE, thereby increasing acetylcholine levels in the brain and enhancing cognitive function through prolonged neurotransmitter availability [67]. However, many of the existing inhibitors have limitations such as adverse side effects, poor bioavailability, and insufficient modulation of acetylcholine levels to achieve a complete therapeutic effect. This has driven ongoing research into the discovery of new AChE inhibitors, particularly those derived from natural sources that may offer better efficacy and safety profiles.

In this regard, Adewusi et al. [16] reported that root extracts of C. mimosoides exhibited significant AChE inhibitory activity. Notably, the organic extract demonstrated the lowest IC₅₀ value (0.03 mg/ml), in contrast to the aqueous extract (0.35 mg/ml), indicating stronger inhibition of the enzyme [16]. The increased activity observed in the organic extract may be due to the greater efficiency of the organic solvents in extracting bioactive compounds with anticholinesterase potential.

Flavonoids, which are abundant in various Chamaecrista species, are natural compounds known for their anticholinesterase properties. Their occurrence in this genus highlights the relevance of further exploration of Chamaecrista as a promising source of new candidates for developing alternative or complementary therapies for AD.

3.2.3. Antioxidant activity

Antioxidants are compounds capable of mitigating oxidative stress by regulating free radicals formation, scavenging reactive species, halting chain reactions, and preventing lipid peroxidation. Free radicals, such as reactive oxygen species and reactive nitrogen species, can damage biomolecules, contributing to the pathogenesis of chronic diseases, including cardiovascular and neurodegenerative disorders, diabetes, and cancer [68]. Given their therapeutic potential, natural antioxidants represent a promising alternative to synthetic compounds, which are often associated with undesirable side effects, making their identification and development of natural antioxidants essential for the prevention and treatment of human diseases and for promoting overall health [69]. The phenolic compounds possess structural features such as hydroxyl groups capable of donating hydrogen atoms, which are key to neutralizing free radicals by cleaving the O–H bond, thereby explaining their high antioxidant capacity [70,71].

The seed oil of C. absus exhibited remarkable free radical scavenging capacity with an IC₅₀ value of 16.78 μg/ml, suggesting the presence of hydrogen-donating constituents capable of efficiently neutralizing DPPH radicals. A study by Reis et al. [72] evaluated the antioxidant activity of C. diphylla using the DPPH method, which revealed significant variations among the different extracts and fractions. The ethyl acetate fraction demonstrated the highest activity, with a CE₅₀ value of 0.11 mg/ml, surpassing the ascorbic acid standard (0.13 mg/ml). The ethanolic extract also exhibited notable activity (CE₅₀: 0.35 mg/ml) [72]. These results correlated with the high content of phenolic compounds, flavonoids, and condensed tannins in both samples.

Similarly, Gomes et al. [25] confirmed the antioxidant potential of C. diphylla, reporting that the ethyl acetate fraction of its leaves had the highest activity (9.44 mmol ET/g) according to the oxygen radical absorbance capacity assay, followed by the methanolic and ethanolic fractions (6.59 and 4.29 mmol ET/g, respectively). The higher antioxidant activity in the fractions than in the crude extract suggests bioactive compound enrichment during the fractionation process [25].

In the case of C. duckeana, the methanolic extract of the stems displayed the highest antioxidant activity, with an IC₅₀ value of 165.71 μg/ml surpassing the standard synthetic antioxidant butylated hydroxytoluene, which had an IC₅₀ of 175.18 μg/ ml [31]. Similarly, the methanolic extract of C. hildebrandtii leaves exhibited potent antioxidant activity (IC₅₀: 8.7 mg/ml), likely due to the synergistic action of various secondary metabolites [32]. The organic extract of C. mimosoides roots showed potent antioxidant activity against 2.2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-Azino-bis(3-ethylbenzothaizoline-6-sulfonic acid) radicals, with low IC₅₀ values of 0.72 and 0.3 mg/ml, respectively [16].

In addition, the radical scavenging activity of the phenolic fractions of C. nictitans was analyzed. The oligomeric fraction demonstrated the highest activity (DE₅₀: 78.6 μg/μmol), while the polymeric fraction showed values similar to the crude extract (DE₅₀: 115.6 and 112.1 μg/μmol, respectively) [35]. In C. repens, the methanolic extract displayed 68.3% antioxidant activity in the β-carotene bleaching assay, whereas its DPPH radical scavenging capacity yielded an IC₅₀ of 2 mg/ ml [73].

Chamaecrista species are promising natural antioxidant sources, primarily attributed to the presence of polyphenolic compounds, particularly flavonoids.

3.2.4. Antiviral activity

Viral infections cause various chronic and acute diseases in humans and animals. These infections are associated with high morbidity and mortality in humans [74]. Current antiviral drugs present several problems, such as high costs, drug resistance, safety concerns, and limitations in efficacy. Viral replication poses a unique challenge, as targeting the virus without harming the host cells is difficult. Viral variability, particularly in RNA viruses, and accumulated genetic mutations contribute to drug resistance, leading researchers to develop new antiviral options [15,35,74]. Emerging and re-emerging viruses represent a significant problem in viral pathogenesis, leading to outbreaks, epidemics, and pandemics. A current example is COVID-19, which is caused by the spread of SARS-CoV-2 and has impacted global social and economic conditions. Therefore, searching for new antiviral agents is crucial, and natural products offer valuable sources of novel chemical compounds with antiviral activity. Numerous preclinical studies have identified natural products with potential in vitro and in vivo antiviral activities, some of which have progressed to clinical trials for drug development [74,75].

However, the antiviral activity of Chamaecrista has not been exhaustively investigated. So far, only C. nictitans has been evaluated for its antiviral properties against herpes simplex virus (HSV ). This virus, which is globally distributed, remains a significant public health issue, infecting 45% –98% of the world’s population[74]. Uribe et al. [15] suggest effective antiviral action, as a dose-dependent effect was recorded as the inhibitory effect of the extract. This activity was attributed to the polar components, initially identified in the aqueous methanolic fraction and later in the dichloromethane and butanol fractions, with inhibition of cytopathic effect (CPE) values ranging from 41.41 to 76.56 μg/ml. The crude extract also exhibited antiviral activity, with a CPE value of 168.75 μg/ml.

Mateos-Martín et al. [35] observed that the oligomeric fraction, at a concentration of 68.7 μg/ml, was the most effective among the analyzed fractions. They suggested that this fraction of C. nictitans extract exerts its action through a particularly effective combination of proanthocyanidins, which have two structural characteristics: monohydroxyphenolic structures and type A linkages. These have been associated with antiviral effects, primarily through inhibiting late transcription. C. nictitans exhibits antiviral properties and acts against herpes simplex virus (HSV), which can be attributed to the presence of polyphenolic compounds. Although acyclovir inhibits the secondary transcription of the virus, the extract of this species inhibits two stages of the HSV replication cycle, adsorption, and secondary transcription, exerting its action intracellularly [15,35,74].

These findings suggested that C. nictitans has therapeutic potential. The structural characterization and isolation of biomolecules are crucial for studying their absorption, distribution, metabolism, excretion, and toxicity properties. As a new natural source for preventing and treating viral diseases, further studies are required.

3.2.5. Antitrypanosomal activity

Trypanosomiasis is caused by a protozoan parasite belonging to the genus Trypanosoma. It is primarily responsible for chronic anthroponotic infections in West and Central Africa and can have severe consequences if not treated adequately [69]. Furthermore, it is classified as a neglected infectious disease, primarily affecting populations in developing countries that receive limited attention in the research and development of new treatments despite its significant impact on public health. Treatment options are limited, underscoring the need for further research to improve the therapeutic possibilities [76].

Only one report has documented the anti-trypanosomal properties of this genus. The crude methanolic extract of C. mimosoides leaves showed significant cessation of parasite motility with increased incubation time and extract concentration. Complete cessation of Trypanosoma brucei brucei motility was observed within 25 minutes at the highest concentration evaluated (4 mg/ml) [77]. These findings indicate that the extract of this species possesses notable in vitro antitrypanosomal activity, which could be attributed to its phytoconstituents, such as flavonoids, terpenes, sterols, and polyphenols. This suggests that these compounds represent a promising source for in vivo treatment of trypanosomiasis [77].

3.2.6. Cytotoxicity

Cancer poses a significant challenge to the global public health. Global demographic trends suggest an increase in cancer incidence in the coming decades, with projections indicating more than 20 million new cases annually by 2025 [78]. Chemotherapy remains a cornerstone of clinical cancer treatment. However, this therapeutic strategy is hindered by challenges, such as tumor heterogeneity, side effects, toxicity, and acquired multidrug resistance, which limit its therapeutic efficacy. Consequently, searching for drugs with reduced toxicity and improved efficacy is a critical priority in medical research. Between 1981 and 2019, approximately 25% of all newly approved cancer drugs were derived from natural products, underscoring the immense potential of this rich resource [79].

Lima et al. [31] demonstrated that methanolic extracts of C. duckeana exhibited over 80% inhibition of cell growth in HL60 leukemia cells and less than 58.17% inhibition against SNB19 (central nervous system), HCT116 (human colon), and PC3 (prostate) cell lines. Stems showed notable cytotoxicity with IC₅₀ values of 137.3 and 106.8 μmol/ ml for HL60 and RAJI cells, respectively. These findings highlight the potential of extracts from this plant species for their promising antitumor and cytotoxic activities [31]. The observed cytotoxicity may be attributed to the presence of the sesquiterpene Eupalinolide A in C. duckeana extracts, which has been noted in the literature for its potent cytotoxic activity [79].

However, further preclinical and clinical studies are required to confirm the anticancer effects of C. duckeana. Once the active compounds responsible for anticancer activity have been identified, such studies could explore the antitumor mechanisms of its constituents. These compounds could serve as promising candidates for developing new cancer treatments or for use in complementary therapies alongside conventional approaches.

3.2.7. Acute toxicity

Acute toxicity testing is generally the first step in assessing the toxicity of a substance. It provides crucial information on the health risks associated with short-term exposure to substances such as drugs. Moreover, these studies can identify early signs of potentially serious adverse effects, enabling the implementation of appropriate preventive measures such as dose adjustments. Such research is paramount before using the substance and lays the groundwork for future clinical toxicity studies [80].

Very few toxicity studies have been conducted in the genus Chamaecrista. Among them, Medugu et al. [81] evaluated the acute toxicity of C. mimosoides extract fractions in mice via the intraperitoneal route, obtaining LD50 values >5,000 mg/kg for the butanol fraction and 3,808 mg/kg for the chloroform and ethyl acetate fractions. These results suggest the low toxicity of the evaluated fractions, with LD50 values ≥1,500 mg/kg.

David et al. [73] conducted an acute toxicity assessment of the methanolic extract of C. repens, obtaining an LC50 value of 269.3 μg/ ml in Artemia salina. Extracts with LC50 values above 200 mg/l in the A. salina lethality assay were considered low toxicity.

Continued investigation of the toxicity of natural product extracts, particularly those with pharmacological activity, is essential for developing Natural Product-Based Libraries. These studies play a critical role in comprehensively evaluating the safety profiles of bioactive compounds, thereby ensuring safe and effective drug discovery [82].