Numerous medicinal properties have been attributed to natural plants. Today, medicinal plants are used to formulate pharmaceuticals, healthcare products, food products, chemicals, and cosmetics. These plants are rich sources of vitamins, minerals, and phytochemicals, which support the human body and help combat various microbial and other health-related ailments. Across the globe, indigenous peoples use various herbs and plants for their diverse medicinal properties. For example, harval teas prepared from such medicinal plants, are well known for their antioxidant properties and benefits, including phenolics and fat-soluble vitamins, which play significant roles in human physiology. Besides vitamins and minerals, phenolics also show potential to support the body by acting as reducing agents, hydrogen donors, singlet oxygen quenchers, and metal chelators against various pathogens and diseases [1-3].

Various bioactive compounds, also known as phytochemicals, such as flavonoids, carotenoids, and phenolic compounds, along with tannins, phytoestrogens, phytosterols, and glucosinolates, and have significant medicinal importance, including tumor-inhibitory and anticarcinogenic potential [4,5]. Nowadays, these bioactive compounds, as secondary metabolites in plants, are considered highly important, and a new era of research is underway to explore their anticarcinogenic, antitumor, antioxidant, and wound-healing effects.

Modern techniques used to identify and isolate these bioactive compounds from various medicinal plants are intricate. While different animals, such as guinea pigs and rats, are often used to assess the antioxidant and therapeutic activities of these compounds, these findings are subsequently translated to human applications [5,6]. Consequently, the use of phytochemicals for therapeutic and antioxidant purposes requires their identification, followed by extraction and refinement into drugs with minimal or no side effects, similar to antibiotics [7].

Inhibiting mutagenic reactions in deoxyribonucleic acid (DNA) and inactivating carcinogens represent a new approach to preventing various cancers and are strategies that utilize anticarcinogens and antioxidants. Enzymatic inactivation, scavenging carcinogens, and inhibition of reactive oxygen species are notable mechanisms utilized for this purpose [8].

In modern medicine, many anticarcinogenic compounds are extracted and purified from traditional plants that have been clinically used to treat different diseases worldwide, such as flavopiridol, epipodophyllotoxin, betulinic acids, combretastatin, camptothecin, silvestrol, taxols, vinblastine, vincristine, irinotecan, and topotecan, which have shown promising results in various studies [9].

Ellagic and gallic acids, as phenolic compounds, can interrupt tumor cell spread and possess DNA-binding properties that effectively interfere with carcinogenic cell development [10]. Different plant parts, such as fruits, shoots, leaves, and roots, from species including Amaranthus gangeticus and Tradescantia zebrina, contain phenolic compounds that have significant anticarcinogenic potential [7].

Few studies have quantified the bioactive composition of plant leaves [11-14]. Comprehensive characterization, functionalization, and utilization studies of bioactive compounds from plant leaves remain scarce, as do studies using spectrophotometric methods as preliminary practical approaches for the screening, identification, and quantification of major bioactive compounds in plant leaves. Plant leaves have complex, heterogeneous matrices that lead to variable levels of pigments, phenolics, and other phytochemicals. For the precise identification and quantification of the individual bioactive compounds, high-performance liquid chromatography (HPLC) or mass spectrometry can be used; however, these techniques are expensive, time-consuming, require specialized solvents, and are not easily scalable for large sample sets. In contrast, UV-Vis spectrophotometry is simple, fast, cost-effective, and widely available in routine laboratories, supporting experimental replication. These experiments also enable simultaneous processing of multiple leaf samples, making them ideal for comparative screening across numerous species, such as those included in this study (24 plants). Moreover, spectrophotometric assays such as the Folin–Ciocalteu assay for total phenolic content, the aluminum chloride assay for flavonoid content, and specific, fixed-wavelength measurements for carotenoids are well-established, replicable, reproducible, and have been widely validated for leaf tissues [4, 15-16]. For this reason, these methods are highly efficient as first-line tools for identifying promising leaf sources of bioactive compounds before more targeted and expensive analyses are undertaken.

This study focuses solely on the identification and quantification of the major bioactive compounds present in traditional medicinal plants widely available in Bangladesh. Variability in extraction systems—such as alkaline or enzymatic extraction—along with differences in sample preparation, yield calculation, and characterization methods, has been identified as a potential source of error affecting the accuracy and comparability of findings across studies. The identification of these bioactive compounds highlights the medicinal value of these traditional plants and their potential health benefits, introducing a new dimension to research and industrial utilization.

2.1. Location of the Plant Samples

A total of 24 plants were randomly selected and collected from the National Botanic Garden (23°48′ N, 90°21′ E) in Mirpur, Dhaka, Bangladesh, during the summer season (March–April) in 2023; their names are listed in Table S1 [17-40]. The National Botanic Garden is maintained by the government organization of the People’s Republic of Bangladesh, where plants are managed using standard horticultural practices without chemical fertilizers. The National Botanic Garden is a vast managed horticultural collection rather than an experimental field or laboratory; therefore, all plants, trees, and shrubs were maintained under routine garden management practices without specific pesticide applications or intentional chemical stress treatments. No species-specific records of soil properties, irrigation schedules, or nutrient management were used because the plants were not cultivated; rather, they were collected as part of a controlled agronomic library and provided experimental samples for researchers in Bangladesh and worldwide. Therefore, no specific cultivation records were available for individual species, as the plants were grown under natural conditions in open beds with regular irrigation. In this study, all plants were selected based on their local medicinal use in Bangladesh and nationwide availability as mature, healthy plants free from visible symptoms of disease, insect infestation, mechanical injury, or physiological disorders. For each species in this study, plant parts (leaves) were collected from three to five individual plants. The leaves were comparable in size and developmental stage and were collected during the same phenological stage. The leaves were fully expanded and mature and were harvested from the middle canopy to ensure that all samples represented the same physiological maturity stage, thereby reducing variation associated with leaf age and development. The leaves were collected between 8:00 and 10:00 AM to minimize variations in metabolite content caused by daylight exposure. After collection, the leafy parts of each plant were thoroughly washed and rinsed multiple times with tap water, and then cut into smaller pieces to obtain a uniform composite sample.

2.1.1. Sample Preparation and Basis of Expression

All collected leaves were washed, cut into small pieces, and homogenized in their fresh state without drying. Bioactive compound contents are expressed as mg per 100 g of fresh weight. Fresh samples were used to avoid degradation of heat-labile compounds such as anthocyanins, lycopene, and β-carotene during drying, and to reflect the bioactive composition as consumed or initially extracted in traditional preparations.

2.2. Extraction of β-Carotene

Five grams of plant leaves were combined with 100 mL of methanol, stirred for 24 h at 25 °C and 150 rpm, and then filtered using Whatman no. 4 filter paper. The methanolic extract was collected, and 100 mg of the extract was subsequently mixed with a combination of 10 mL acetone-hexane (4:6). After vigorously shaking the mixture for one minute, it was filtered again using Whatman filter paper no. 4 [41]. A T80 UV/Vis Spectrophotometer (PG Instruments, Leicestershire, UK) was used to measure absorbance. Carotenoid absorbance was measured at 453 nm, 505 nm, and 663 nm, and carotenoid content was calculated using Equation (1) and expressed as mg β-carotene equivalents per 100 g fresh weight.

2.3. Extraction of Lycopene

Colorimetric determination of lycopene from plant leaves was performed by extraction with hexane, ethanol, and acetone, followed by absorbance measurement at 503 nm. 1 g of plant leaves was collected and soaked in 80 mL of a hexane-ethanol-acetone mixture (2:1:1) in a flask. Then, the solution was vortexed for 10 min, 10 mL of water was added, and the mixture was vortexed for a further 10 min [15]. Lycopene absorbance was measured at 503 nm and expressed as mg per 100 g fresh weight.

2.4. Extraction of Flavonoids

Flavonoid extract was prepared using methanol: 10 mg of plant leaves were soaked in 10 mL of methanol, and 1 mL was collected in a test tube. 5 mL of water and 0.1 mL of potassium acetate (CH₃CO₂K) were added to the test tube, which was then left to rest for 5 min. Then, 3 mL of aluminum chloride (AlCl₃) was added, and the mixture was allowed to stand for 30 min at room temperature[4]. QC samples for accuracy determination at six concentrations (0, 10, 20, 30, 40, and 50 µg/mL) were also prepared by serial dilution of the freshly prepared rutin stock solution and used within 24 h. Flavonoid absorbances were measured at 415 nm, using equation 2 to determine TFC, where Y is the absorbance of the sample, and X is the total flavonoid content in mg/100 g Rutin equivalents (RE) per 100 g fresh weight.

2.5. Extraction of Anthocyanin

1 g of plant samples and 40 mL of 50% ethanol were placed in a flask at pH 3.0, then kept at a warm temperature (50 °C) for 1 h. The solution was then filtered, and a 1 mL aliquot was collected in a test tube and combined with 4 mL of methanol [4]. Anthocyanin absorbance was measured at 530 nm and calculated using the following equation. Anthocyanin content was expressed as mg cyanidin-3-glucoside equivalents per 100 g fresh weight.

2.6. Extraction of Total Phenol

This method used 25% ethanol for extracting total phenols from plant leaves. One gram of plant leaves was soaked in 20 mL of 25% ethanol for 15 min. The solution was then filtered through Whatman No. 2 filter paper, and a 1 mL sample was collected from the filtrate, mixed with 5 mL Na₂CO₃ and 1 mL Folin reagent, and transferred to a volumetric flask. Then it was kept at room temperature for 1 h [4, 16, 42]. Quality control (QC) samples to assess accuracy at five concentrations (10, 20, 30, 50, and 60 PPM) were also prepared by serial dilution of the freshly prepared gallic acid stock solution, which was used within 24 h. Phenolic content was quantified at 765 nm using spectrophotometry (T80 UV/Vis Spectrophotometer, PG Instruments, UK) by comparing absorbance values with a gallic acid calibration curve and expressed as milligrams of gallic acid equivalents per 100 g fresh weight(mg GAE/100 g fresh weight). Equation 2 was used to calculate the total phenolic content (TPC), where Y is the absorbance of the sample, and X is the total phenolic content, expressed as mg GAE/100 g fresh weight.

2.7. Statistical Analysis

All measurements were performed in triplicate for each sample, and averages and standard deviations were calculated from these measurements. Data were analyzed using R statistical software (Windows version 4.3.3) and figure are generated in GraphPad Prism (Version 8.3.0). A single-factor analysis of variance was conducted. Duncan’s Multiple Range Test (DMRT) was used to identify significant differences, with differences considered significant at p ≤ 0.05.

3.1. β-Carotene Content

Figure 1 and Table S2 present the concentrations of extracted β-carotene from plant parts, revealing significant amounts in the respective species, in which Ipomoea batatas showed the maximum concentration at 153.21 mg/100 g and Ajuga reptans at 138.67 mg/100 g, indicating a high β-carotene content in the leaves. In addition to Ipomoea batatas and Ajuga reptans, the leaves of Lawsonia inermis and Euphorbia cotinifolia also contained significant amounts of β-carotene, comparable to those found in Amaranthus gangeticus, establishing them as excellent sources of this carotenoid. To maintain a healthy life, a daily intake of 15–45 mg of β-carotene is recommended, as supported by previous nutritional studies [43]. In Bangladesh, Ipomoea batatas and Amaranthus gangeticus leaves are traditionally used in food preparations such as steaming and infusion with oil, whereas Euphorbia cotinifolia is highly toxic, and Lawsonia inermis is prohibited for direct consumption. For pharmaceutical use, all species, including toxic ones, can be processed under controlled environmental conditions into standardized extracts, powders, coated or encapsulated formulations. Additionally, although the practice is uncommon in Bangladesh, young shoots of Ajuga reptans can be utilized in salads in Brazil [44].

β-carotene, which is commonly present alongside water-soluble and fat-soluble vitamins, is abundant in plant leaves and has been reported to inhibit various cancers [44]. The antioxidant capacity of plant leaves varies with their content of metabolites such as vitamins, carotenoids such as β-carotene and lycopene, and other pigments [45].

Carotenoids, which are precursors to vitamin A, are typically derived from flowering plants and herbs and serve as colorants and bioactive compounds; they are very common in tropical plant species, as evidenced by previous studies [46]. Comparable levels of β-carotene have been reported in medicinal plants [11-13]. Still, certain plant leaves, such as wine palm (Mauritia vinifera), are promising sources of β-carotene [47].

3.2. Determination of Lycopene

There are different types of carotenoids, such as lycopene and β-carotene, with lycopene having a nearly two-fold higher capacity to reduce singlet oxygen than β-carotene [48]. Lycopene plays a crucial role in defense against carcinogenic diseases by protecting DNA, lipid particles, low-density lipoproteins, and proteins from free radical attacks [49].

According to Figure 2 and Table S2, leaves of Amaranthus gangeticus exhibited the highest lycopene concentrations at 109.44 mg/100 g, followed by Tradescantia zebrina leaves at 84.11 mg/100 g. Furthermore, leaves from Tradescantia zebrina, Ipomoea batatas, and Lawsonia inermis are significant sources of lycopene [50,51].

3.3. Flavonoids Determination

Figure 3 and Table S2 present the quantification of flavonoid content, showing that Cordyline fruticosa varieties exhibited the highest flavonoid levels, with Cordyline fruticosa at 810.46 mg/100 g RE, Cordyline rubra at 502.13 mg/100 g RE, and Plectranthus scutellarioides at 352.18 mg/100 g RE. The flavonoid content of these plant leaves falls within a range similar to that reported in previous studies on medicinal plants [52-55]. However, the sources of plant leaf samples vary with environmental conditions and soil composition, making direct comparisons challenging.

Notably, samples of Cordyline species exhibited significantly higher flavonoid levels (p < 0.05) than other plant leaves. For instance, 17.1 ± 2.8 mg RE/100 g d.b. for Tradescantia zebrina and 10.6 ± 4.0 mg RE/100 g d.b. for Tradescantia pallida (Malaysia), values which surpass those found in this study [14]. Similarly, 3.93 ± 0.008 mg quercetin/100 g d.b. for Tectona grandis and 27.2 mg RE/100 g d.b. for Prunus cerasifera, values close to those reported in several studies [56,57]. Again, another study reported 6.92 mg RE/100 g d.b. for Oxalis corniculata, while 14.32 mg quercetin/g for Basella alba, both of which are notably lower than the levels observed here [58,59].

3.4. Determination of Anthocyanins

Anthocyanins, vibrant pigments responsible for the blue, red, and purple hues in leaves and fruits, are notably abundant in various berries [60]. Among the species examined, Tradescantia spathacea exhibited the highest level of anthocyanins at 267.18 mg/100 g, followed by Oxalis triangularis at 161.67 mg/100 g and Alternanthera dentata at 151 mg/100 g (Figure 4 and Table S2). With their diverse colors and potential health benefits, anthocyanins are used in dietary supplements, nutraceuticals, and pharmaceutical products.

The total anthocyanin contents observed in the leaves of Tradescantia spathacea, Oxalis triangularis, and Alternanthera dentata were lower than those reported for various vegetable leaves. For instance, several studies have reported that purple cabbage leaves contain 629 mg/100 g d.b., red lettuce varieties contain 100 to 300 mg/100 g d.b., and purple leaves of Perilla frutescens contain 42.33 mg/100 g d.b. [61-63].

3.5. Total Phenolic Determination

Previous studies have categorized the phenolic content of plant leaves into three groups: low, moderate, and high. Generally, plants with phenol content below 500 mg GAE/100 g are classified as having low concentrations of phenolic compounds, those ranging from 500 to 2500 mg GAE/100 g fall into the medium concentration category, and those above 2500 mg GAE/100 g are categorized as having high concentrations of phenolic compounds [64].

Tradescantia zebrina exhibited the highest level of phenolic compounds among the plants examined, followed by Lawsonia inermis, Plectranthus scutellarioides, and Acalypha wilkesiana (Figure 5 and Table S2). The leaves of these plants contain high levels of phenolic compounds, making them excellent sources. Except for Caladium Bombshell, the leaves of the other examined plants can be categorized as having medium levels of phenolic compounds and may still represent good sources of these compounds.

Notably, higher levels of phenolic compounds were found in Tectona grandis, Tradescantia zebrina, and Tradescantia pallida (Malaysia) than in this study [14,57]. However, the levels reported for Prunus cerasifera were comparable to those obtained in several studies [56]. Previous studies reported lower levels in Oxalis corniculata and Basella alba [58,59]. These discrepancies may be attributed to various factors affecting the properties of bioactive compounds in plant leaves, such as cultivation methods, climate, plant varieties, and the time of year of harvest [65].

3.6. Correlation Analysis

Pearson correlation analysis was performed using Prism to examine the relationships among the phytochemical components (β-carotene, lycopene, flavonoids, anthocyanins, and total phenolic content) in the plant species under investigation (Figure 6). To calculate the correlation coefficient (r) and its 95% confidence interval (CI), we used a metric to assess the relationship between each pair of variables. The correlation coefficient ranges from −1 to +1, where values close to +1 indicate strong positive relationships, while values close to −1 indicate strong negative relationships.

In this study, several variable pairs showed extremely strong positive correlations, indicating that their concentrations tend to vary in proportion to each other. For instance, the correlation between β-carotene and lycopene was strongly positive (r = 0.9264) and had a 95% CI of 0.8141 to 0.9264 because they share a similar biosynthetic pathway as carotenoids. In addition, flavonoids and total phenolic content showed moderately strong positive correlations, as expected due to the fact that flavonoids are a subset of total phenolic content.

Furthermore, several pairs exhibited strong positive correlations, indicating that the concentrations of these phytochemicals tended to increase together. In particular, anthocyanins and total phenolic content showed a very strong positive correlation (r = 0.9712), suggesting that plant species with higher anthocyanin concentrations also possessed higher total phenolic contents.

Most remaining variable pairs showed very weak or negligible correlations, indicating no significant relationship; for instance, anthocyanins and flavonoids had a very weak correlation (r = 0.01494, 95% CI: 0.02131 to 0.9919). Confidence intervals (95%) for the correlation coefficient indicate the reliability of the estimates: wide confidence intervals (0.8141 to 0.9264) indicate greater uncertainty in the correlation estimate, whereas narrow confidence intervals (0.5491 to 0.9977) indicate a stronger estimate. Correlations between variables whose confidence intervals include zero were not statistically significant.

This information on the studied correlations holds significant value regarding different metabolic pathways of the studied bioactive compounds because, for instance, a positive correlation of β-carotene and lycopene suggests common regulation, as carotenoids are theoretically controlled by the same genetic and environmental factors, while a lack of correlation between anthocyanins and flavonoids may be interpreted as their separate control. Again, because of resource allocation at the biosynthetic level, the strong positive correlation between anthocyanins and total phenolic content suggests that these phytochemicals accumulate concurrently in the studied ornamental plant species. This finding is biologically plausible because anthocyanins are a subclass of phenolic compounds and share common biosynthetic pathways. Therefore, increased anthocyanin accumulation is often associated with increased total phenolic content.

3.7. Practical Implications and Beneficiaries

This study provides a detailed and reliable dataset on the bioactive compound profiles of 24 medicinal plant species commonly found in Bangladesh, which may benefit several groups within the research and industrial communities. First, researchers can use this dataset as a baseline for further investigations into phytochemicals across different extraction methods, characterization, bioactivity evaluation, compound isolation, and the development of nutraceuticals, functional foods, and pharmaceutical products. Identifying plant species with varying concentrations of bioactive compounds may also provide promising candidates for advanced analytical and chemical studies using metabolomic and chromatographic techniques.

Second, the food, pharmaceutical, and nutraceutical industries may use these findings to identify economically valuable plant species as potential natural sources of antioxidants, phenolic compounds, flavonoids, anthocyanins, β-carotene, and lycopene, which may facilitate the extraction and development of natural ingredients, functional food products, dietary supplements, and plant-derived therapeutic innovative formulations.

Third, government organizations such as policymakers, different public and private agricultural extension agencies, and local and international botanical conservation authorities may use the results to prioritize the conservation, sustainable utilization, and cultivation of medicinal plant species with high bioactive compound potential. These findings also support future strategies to promote value-added utilization of indigenous plant species and strengthen the medicinal plant sector.

Finally, this study can contribute to sustainable development awareness by encouraging the use of locally grown and available medicinal plants as renewable natural resources to produce high-value bioactive compounds that are highly effective for the treatment of chronic diseases. This contribution not only potentially reduces the dependence on synthetic medicine and additives but also promotes environmentally sustainable resource utilization and creates new economic opportunities through the development of plant-based health products.

Different plant parts contain various pigments, such as β–carotene, lycopene, anthocyanins, and flavonoids, supporting the value of utilizing these plant species, given their significant amounts of bioactive compounds and diverse macro- and micronutrients, particularly anthocyanins, flavonoids, and phenolic compounds. This study provides valuable evidence confirming the nutritional properties of medicinal plant leaves, particularly the diverse properties of identified pigments and compounds, which are not only rich sources of bioactive compounds but also can be utilized as low-cost, renewable resources for the pharmaceutical industry, thereby providing positive economic and environmental impacts. From a traditional preparation perspective, Ipomoea batatas and Amaranthus gangeticus leaves are locally processed through light steaming or infusion in Bangladesh, making them accessible sources of bioactive compounds and vitamins for local populations. For pharmaceutical and nutraceutical applications, all studied species, including Tradescantia zebrina, Cordyline fruticosa, Lawsonia inermis, and even the toxic Euphorbia cotinifolia, can be processed into standardized extracts, powders, or encapsulated formulations through controlled chemical, physical, or mechanical extraction methods. These findings confirm that bioactive compounds may play significant roles in protecting against various diseases, suggesting broader applications beyond their use as sources of medicine.

This study did not involve controlled cultivation because, in Bangladesh, dedicated cultivation practices do not exist for these plants, except for Amaranthus gangeticus and Ipomoea batatas. Consequently, the plant materials were collected from a managed botanical garden rather than a controlled cultivation experiment. No specific agronomic variables or cultivation parameters, such as soil nutrient composition, irrigation schedules, and fertilizer history, were available for individual plant species; therefore, these factors could not be standardized. Although these factors can influence bioactive profiles, future studies should build on this work by conducting controlled cultivation experiments to separate genotypic effects from environmental influences on bioactive compound accumulation.

Methodology, investigation, formal analysis, data curation, writing—original draft preparation: T.K; Conceptualization, validation, software, resources, writing—review & editing, visualization, supervision, and project administration: M.A. All authors have read and agreed to the published version of the manuscript.

Data are available from the corresponding author upon reasonable request.

Not applicable. This study did not involve human participants or animal subjects; therefore, ethical approval was not required. The plant leaf samples were collected from the National Botanic Garden (Mirpur, Dhaka, Bangladesh) in accordance with the garden’s regulations and relevant national guidelines, and no specific collection permits were required for the small quantities of plant material used.

The authors declare no conflicts of interest.

The study did not receive any external funding and was conducted using only institutional resources.

The authors would like to acknowledge the support and facilities provided by the Laboratory of Food Processing and Technology, Department of Food Engineering and Nutrition Science, State University of Bangladesh, where this study was conducted. EndNote was used for reference management and citation formatting during the preparation of this manuscript.

Download Supplementary Tables S1 and S2 here.

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