Optimization of Solvent Extraction for Phenolic, Flavonoid, and Elemental Profiling of Nerium oleander, Bulbine frutescens, and Cotyledon orbiculata: Implications for Phytomedicinal Applications

1. Introduction

Medicinal plants continue to play a significant role in primary healthcare owing to their rich diversity of bioactive phytochemicals and essential nutrients. Among the most important classes of phytochemicals are phenolic compounds and flavonoids, which are recognized for their potent antioxidant, anti-inflammatory, antimicrobial, and wound-healing properties [1,2]. Phenolic compounds act as effective free-radical scavengers, thereby reducing oxidative stress and protecting biological systems from cellular damage associated with chronic diseases and impaired tissue repair [3,4]. Flavonoids, a major subclass of phenolic compounds, contribute to therapeutic efficacy through multiple mechanisms, including modulation of inflammatory pathways, inhibition of microbial growth, enhancement of collagen synthesis, and protection against oxidative damage [5,6]. Consequently, the determination of total phenolic and flavonoid contents is widely employed as an indicator of the medicinal potential and quality of plant extracts.

In addition to phytochemicals, medicinal plants contain essential macro- and microelements that contribute to their nutritional and therapeutic value. Elements such as calcium, magnesium, potassium, iron, zinc, manganese, and copper are involved in numerous physiological processes, including enzyme activation, immune function, antioxidant defence, cellular metabolism, and tissue regeneration [7]. The presence of these elements may enhance the biological activity of plant extracts through synergistic interactions with secondary metabolites, particularly in applications related to wound healing and inflammation management. Furthermore, elemental profiling serves as an important quality-control measure by assessing nutritional value and ensuring the absence of potentially toxic elements [8].

Nerium oleander, Bulbine frutescens, and Cotyledon orbiculata are medicinal plants traditionally used for the treatment of wounds, skin disorders, inflammation, and various infectious conditions. Previous studies have attributed these therapeutic effects to the presence of phenolic compounds, flavonoids, and other bioactive metabolites. For example, Nerium leaves were found to be rich in carbohydrates, flavonoids, alkaloids, steroids, cardiac glycosides and tannins [9]. It has been demonstrated that Bulbine frutescens species contain chemotaxonomic biomarkers, responsible for enhancing skin health and wound healing [10]. Phytochemical analysis of C. orbiculata leaf extract has also confirmed the presence of cardiac glycosides, flavonoids, phenolics, reducing sugars, saponins, condensed tannin, gallotannin, and triterpene steroids [11]. However, comparative information regarding the recovery of these constituents under different extraction conditions and their relationship with elemental composition remains limited. Therefore, the evaluation of total phenolic content, total flavonoid content, and elemental nutrients in these medicinal species is essential for understanding their phytochemical characteristics, validating their traditional uses, and supporting the development of standardized phytotherapeutic products. Given the significant contribution of phenolic compounds, flavonoids, and mineral nutrients to the biological activities and nutritional value of medicinal plants, their quantitative evaluation provides important insights into the therapeutic potential and quality of plant-derived products. Therefore, this study aimed to determine the total phenolic content, total flavonoid content, and elemental composition of Nerium oleander, Bulbine frutescens, and Cotyledon orbiculata extracts and to evaluate the influence of extraction conditions on the recovery of these bioactive constituents.

2. Materials and Methods

2.1. Plant Material Collection and Preparation

The plant material was identified by a traditional healer and was purchased at Magic Lawns Nursery in Vereeniging, Gauteng Province, South Africa (26°40′S, 27°55′E). To comply with botanical documentation and alignment with scientific standards, the plant name was cross-checked and validated using internationally recognized taxonomic databases, including Plants of the World Online (POWO), World Flora Online (WFO), and the Medicinal Plant Names Services (MPNS). Leaves were thoroughly washed with distilled water, air-dried at ambient temperature (25 ± 2°C) for 14 days, and ground to a fine powder (particle size < 0.5 mm) using a laboratory mill. The powdered material was stored in airtight containers at 4°C until further analysis.

2.2. Plant Material Extraction

Powdered leaf material (50 g) was extracted with 500 mL of each solvent (1:10 w/v ratio) by maceration at room temperature (25 ± 2°C) with intermittent shaking (150 rpm) for 72 hours. The solvents evaluated included water, methanol, ethanol, acetone, dichloromethane, chloroform, ethyl acetate, and hexane, selected to encompass a broad polarity range (polarity index: 0.1–9.0). The extract was filtered through Whatman No. 1 filter paper, and the residue was re-extracted twice with fresh solvent (2 × 250 mL) to ensure complete extraction. The combined filtrates were concentrated under reduced pressure at 40°C using a rotary evaporator (Büchi Rotavapor R-210) to obtain the crude extract, which was then dried to constant weight in a vacuum desiccator and stored at 4°C until analysis. All extractions were performed in triplicate, and results are expressed as mean ± standard deviation.

2.3. Spectrophotometric Analysis of Total Phenols using Garlic Acid Standard

Total phenolic content of extracts was determined according to the modified method of Khatiwora [12]

2.3.1. Preparation of extract Solution

1 mg of extract was dissolved in 5 ml of its extracting solvent.

2.3.2. Preparation of garlic Acid Standard Stock Solution

About 5 mg of garlic acid was dissolved in 10 ml of ethanol, and then further diluted to 100 ml with deionized water in a volumetric flask. Different concentrations (0.005 – 0.05 mg/ml) were prepared from the standard stock solution.

2.3.3. Preparation of Forlin-Ciocalteu Reagent (FCR)

About 2 ml of FCR was diluted 10x with deionized water.

2.3.4. Preparation of 7.5% sodium carbonate solution

About 7.5 g of sodium carbonate was dissolved in 100 ml of deionized water, and the pH was adjusted to 10.

2.3.5. Preparation of blank Solution

1 ml of ethanol, 5 ml of FCR and 4 ml of sodium carbonate solution were mixed.

2.3.6. Procedure

To 1 ml of standard/extract, 5 ml FCR and 4 ml sodium carbonate solutions were added. The mixtures were incubated at 25 ◦C for 45 min. Absorbance at 760 nm were measured using UV-VIS spectrophotometer. A standard calibration curve of concentration vs. absorbance was plotted, from which total phenolic content of extracts was expressed as mg of garlic acid equivalents.

2.4. Spectrophotometric Analysis of Flavonoids using Quercetin Standard

Total phenolic content of extracts was determined according to the modified method of Ramos [13].

2.4.1. Preparation of Extract Solution

1 mg of extract was dissolved in 5 ml of its solvent.

2.4.2. Preparation of quercetin Standard Stock Solution

About 5 mg of quercetin was dissolved in 10 ml of ethanol, and then further diluted to 100 ml with deionized water in a volumetric flask. Different concentrations (0.005 – 0.05 mg/ml) were prepared from the standard stock solution.

2.4.3. Preparation of 5% aluminium Chloride Solution

About 5 g of aluminium chloride was dissolved in 100 ml of deionized water.

2.4.4. Procedure

To 6 ml of standard/extract solutions, 2 ml of 5% aluminium chloride was added, and the volume was made up to 25 ml with deionized water. The mixtures were incubated at 25 ◦C for 35 min. The wavelength of maximum absorption was determined using UV-VIS spectrophotometer, from which the absorbance was measured. A standard calibration curve of concentration vs. absorbance was plotted, from which total phenolic content of extracts was expressed as mg of garlic acid equivalents.

2.5. Elemental Analysis of Plant Extracts Using ICP-OES Method

2.5.1. Reagents Preparation

All solvents and reagents used were of analytical grade. Distilled water was used as a solvent for preparation of solutions. Saturated solutions of extracts were prepared by dissolving 1 mg of extract in 1 ml of distilled water. Stock solutions of standards were prepared from the multi- element solution, and different dilutions were prepared from which the calibration curve was plotted.

2.5.2. Analytical Procedure

The Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) method was used for the elemental analysis of elements in the extracts.

2.6. Data Analysis

Results were expressed as mean ± standard deviation. Comparative evaluation was based on: Sensitivity, accuracy and reproducibility.

3. Results

3.1. Determination of total Phenols, Flavonoids and Elemental Nutrients from Nerium oleander (N. oleander), Bulbine frutescens (B. frutescens, and Cotyledon orbiculata (C. orbiculata)

3.1.1. Spectrophotometric Determination of Total Phenolic and Flavonoid Content Using Gallic Acid and quercetin Standards, Respectively

• Figure 1 is the calibration curve of the Gallic acid standard;
• Table 1 shows the results of the absorbance versus the concentration of the Gallic acid standard;
• Figure 2 is the calibration curve of the quercetin standard;
• Table 2 shows the results of the absorbance versus the concentration of the quercetin standard;
• There was good correlation between absorbance and concentration of both Gallic acid and quercetin, with correlation coefficients of 0.99 and 0.993, respectively.

3.1.2. Total Phenolic Content of N. oleander, B. frutescens, and C. orbiculta Extracts Expressed as Garlic Acid Equivalent

• Figure 3 is the graphical representation of the total phenolic content of N. oleander, B. frutescens, and C. orbiculata;
• Table 3 shows the results of the total phenolic content of N. oleander, B. frutescens, and C. orbiculta extracts;
• The total phenolic content in N.oleander and B.frutescens increased with decrease in polarity of the solvent from water to dichloromethane, and decreased from dichloromethane to ethyl acetate;
• Dichloromethane resulted in the highest total phenolic content of 6.936±0.613, 0.798±0.13, and 1.061±0.587 mg Gallic Acid Equivalent (GAE)/ 100 mg sample for N. oleander, B. frutescens and C. orbiculata, respectively;
• There seemed to be no significant differences in the total phenolic content between B. frutescens and C. orbiculata.

3.1.3. Total Flavonoid Content of N. oleander, B. frutescens, and C. orbiculta extracts expressed as quercetin equivalent

• Figure 4 is the graphical representation of the total flavonoid content of N. oleander, B. frutescens, and C. orbiculata;
• Table 4 shows the results of the total flavonoid content of N. oleander, B. frutescens, and C. orbiculta extracts;
• Hexane extract of N. oleander resulted in the highest total flavonoid content of 8.793±0.02 Querceting Equivalent (QE)/ 100 mg sample, respectively;
• Methanol extracts of both B.frutescens and C.orbiculata had the highest flavonoid content of 1.625±0.022 and 1.157±0.074 mg Quercetin Equivalent (QE)/ 100 mg sample, respectively;
• Generally, N.oleander presented the highest flavonoid content of the three plants;
• There seemed to be significant difference in the flavonoid content of all the three plants.

3.1.4. Investigating Whether or Not There’s Any Significant Differences in the Total Phenolic Content Between B. frutescens and C. orbiculata

• Table 5 illustrates the descriptive statistics of the B. frutescens and C. orbiculata data to evaluate if the data followed normal distribution. For normal distribution, the mean and median should be similar. The skewness of the data points should be closer to zero. There was no conclusive evidence from the descriptive statistics to suggest if the data was normally distributed;
• Figure 5 shows the Q-Q plot of the B. frutescens and C. orbiculata data to further investigate the normality of the data. It was evident that the data was normally distributed since most data points were lying in or near the straight line, and thus ANOVA and Tukey’s HSD tests were appropriate to determine the significant difference of the phenolic content between B. frutescens and C. orbiculata;
• Table 6 displays the results for a two-way ANAVO test with replication, whilst Figure 6 a graphical illustration of the similarities or differences in the phenolic content using Tukey’s HSD test;
• The plant type had no significant effect (F = 1.64, p < 0.05), indicating that both plants had similar amounts of total phenolic content (Table 6);
• The analysis showed a significant effect of extraction solvent (F = 15.15, p < 0.05), demonstrating that solvent type played a critical role in the extraction efficiency of phenolic compounds from the same plant type (Table 6);
• There was a significant interaction effect between extracting solvent and the type of plant (F = 4.42, p < 0.05), suggesting that the effectiveness of extracting phenolic compounds from a specific type of plant depends on the solvent employed (Table 6). For example, both methanol and ethyl acetate extracted different amounts of total phenolic compounds from B. frutescens and C. orbiculata (Figure 6).

3.1.5. Determination of Macro and Trace Elements from Nerium Oleander Plant Extracts Using ICP-OES

• Table 7 gives the results for the elemental nutrients of a multi element standard using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Figure 7, Figure 8, Figure 9 and Figure 10 are the calibration curves generated from the data in Table 7;
• The calibration curves show good correlation between elemental nutrients and the intensity;
• Table 8 and Table 9 display the results of the macro and micro/trace elemental nutrients for the water, methanol, acetone, chloroform, ethyl acetate, and hexane extracts of Nerium oleander, calculated from the multi elements standard calibration curves;
• Figure 11 is the graphical illustration of the macro and micro/trace elemental nutrients of the Nerium oleander water, methanol, acetone, ethyl acetate, and hexane extracts;
• Methanol had the highest concentration of calcium and magnesium, which were 1.42 and 1.98 mg/L respectively.
• Ethyl acetate displayed the highest concentration of 0.96 mg/L for sodium, whilst hexane had the highest concentration of potassium, at 2.88 mg/L;
• Chromium, copper, nickel and lead were not present or their concentration were below detectable limits from all the extracts;
• The highest manganese and zinc concentrations of 0.26 and 0.2 mg/L were found in the water extract;
• The highest iron concentration of 0.03 mg/L was found in both water and acetone extracts.

3.2. Formatting of Mathematical Components

The phenolic and flavonoid content of samples in µg/mL was calculated using a linear equation,

$$x={\displaystyle \frac{1000}{m}}(y+b)$$

(1)

where “y” is absorbance of samples; “x” is concentration of samples; “m” is the gradient of the graph, and “b” is the constant in the equation (see Figure 1 and Figure 2).

The phenolic and flavonoid content of samples in mg/100 mg sample was calculated using equation,

$$concentration=x\times dilFactor\times {\displaystyle \frac{massofextract\left(seeTable7\right)}{massofrawmaterialextracted\left(seeTable7\right)}}$$

(2)

where

dilFactor = dilution factor (see 2.2.1);

The elemental nutrient content of samples in mg/L was calculated from a quadratic equation,

$$y=a{x}^2+bx+c$$

(3)

where

$$x=-\sqrt{{\displaystyle \frac{1}{a}}\left(y-c\right)+{\left({\displaystyle \frac{b}{2a}}\right)}^2}-{\displaystyle \frac{b}{2a}}$$

and “x” and “y” are the concentration and “intensity”, respectively.

4. Discussion

Bhuvaneshwari et al. [14], reported negative results for the presence of total phenols and wflavonoids in Nerium oleander (N. oleander). However, Motaung et al. [15] reported positive results for the presence of phenols and flavonoids. In the same study by Motaung and company, phytochemical screening of B. frutescens reported negative results for phenols, whilst positive results were reported for the acetone and hexane extracts of C. orbiculata. They reported positive results for the presence of total flavonoids for C. orbiculata.

Mohadjerani reported the phenolic content of 4.54 ± 0.23 µg/mL for water extract of N. oleander leaves [16]. The phenolic content for the water extract of N. oleander leaves obtained in this study was 1.061±0.587 mg Gallic acid (GAE)/ 100 mg sample. Ali et al. reported the total phenolic content of Nerium oleander flower to be 13.654±3.32 mg GAE/100 g essential oil [17], which suggests that most of the phenolic compounds are contained in the flower part. Teffo et al. reported a total phenolic content of 83.14 ± 53.4 mg GAE/100 mg sample for B. frutescens [18], which is higher than 0.798±0.13 mg GAE/100 mg sample reported in this study. They reported a total phenolic content of 1.70 mg GAE per gram dry matter for C. orbiculata, which is similar to 1.061±0.587 mg GAE/100 mg dry matter reported in this study.

The highest flavonoid content of 8.793±0.02 mg QE/ 100 mg sample was obtained by the hexane extract. Redha obtained the flavonoid content of 2.5 ± 0.04 and 3.287 ± 0.10 mg Rutin Equivalents (RE) per 100 gram sample for water and methanol extracts of N. oleander leaves, respectively [19]. The flavonoid content from the flower were 7.10 ± 0.02 and 40.04 ± 0.09 mg RE per gram sample for water and methanol extracts of N.oleander, respectively. The flavonoid content for water and methanol in this present study were 4.139±0.078 mg QE/100 mg sample and 7.796±0.055 mg Quercetin Equivalent (QE)/100 mg sample, respectively.

Methanol extracts of B. frutescens and C. orbiculata resulted in the highest flavonoid contents of 1.625±0.022 and 1.157±0.074 mg QE/100 mg sample, respectively. Shikalepo and company reported the flavonoid content of B. frutescens to be 7.160 ± 0.05 mg QE/100 mg extract [20]. Aremu et al. reported the flavonoid content of 0.008 µg Catechin Equivalent (CTE)/g dry matter of C. orbiculata [21].

Adamu and company indicated that calcium is necessary for normal functioning of cardiac muscles, regulation of cell permeability, blood coagulation [22]. Excess of calcium in blood results in calcification of several internal organs. Deficiency of calcium causes diseases like rickets, osteoporosis [22]. Magnesium is important to all the cells in humans, and is present in many enzymes involved in proteins, lipids, and carbohydrate metabolism. In plants, Magnesium is present in chlorophylls. Magnesium is required in the plasma and extracellular fluid, where it helps maintain osmotic equilibrium [22].

Sodium just like Potassium, is also one of the major electrolytes in the blood and without sodium the body cannot be hydrated, it would dry off [22]. They recommended that too much of sodium may cause the cell to break down. Sodium is of great importance for the regulation of many systems in the body. They advised that potassium helps in the proper function of brain and nerves, and thus it helps in prevention of stroke. They also indicated that potassium regulates acid-base and water balance in the blood and tissues, and suggested that high potassium in diet lowered blood pressure in individuals with raised blood pressure [22]. They emphasised that potassium is essential in protein biosynthesis by ribosomes. Mogos advised that potassium is also an essential macro-element for a human as it is involved in muscle contraction, in lipids metabolism, in proteins synthesis, maintaining the fluid and electrolyte balance in the body and is responsible in the nerve impulses sending [23].

Chromium, copper, nickel and lead were not detected from all the extracts. Chromium is known to be essential in the synthesis of fatty acids and cholesterol, but excess results in asthma, shortness of breath, liver and kidney damage and allergic reactions [23]. Zayed and Terry suggested that since chromium was highly toxic, carcinogenic and is lethal at high dosage, it is required by the human body in very trace amount [24]. The permissible limits of chromium in edible plants set by FAO/WHO was 0.02 ppm [25]. Copper is regarded as an essential redox-active transition element that is critical in various metabolic processes. They also found to be essential to the human body since it forms a component in many enzyme systems such as cytochrome oxidase, lysyl oxidase and an iron-oxidizing enzyme in the blood [22]. FAO/WHO set the permissible limit for copper in edible plants at 3.00 ppm [26].

Nickel is required for production of insulin, and it is a component of several enzymes i.e. carbon monoxide dehydrogenase, urease, hepatic microsomal enzymes etc. [26]. However, excess of nickel was found to cause allergic dermatitis known as nickel itch, which usually occurs when skin is moist [22]. The permissible limit of nickel in edible plants set by FAO/WHO was 1.63 ppm [26]. Lead is known to have no beneficial effects in humans, and the permissible limit set by FAO/WHO in edible plants was 0.43 ppm [26]. Lead was found to cause both acute and chronic poisoning, and has adverse effects on kidney, liver, vascular and immune system [27,28]. It also causes a rise in blood pressure, miscarriages, subtle abortion, and decline in fertility of men through sperm damage and diminishing abilities of children and disruption of nervous systems [29].

The highest manganese concentration of 0.26 mg/L was found in the water extract. Manganese was found to be essential for the development of normal structure, reproduction, metabolism of amino acids, lipids, carbohydrates and functioning of central nervous system, and its deficiency was found to results in tissue damage. However, excess manganese was also found to be the cause of pneumonia, affecting the reproductive system, which may lead to infertility [22]. FAO/WHO set the manganese limit in edible plants at 2.00 ppm [26]. Zinc is one of the most important minerals in the body, but high concentration of zinc was found to be neurotoxin [30]. The permissible limit set by FAO/WHO for zinc is 27.4 ppm [26]. Zinc was found to be a co-factor for enzymes in the body. It also takes part in synthesis of DNA, proteins and in insulin biosynthesis, storage, and secretion [31]. In this study, the water extract had the highest zinc concentration of 0.2 mg/L.

Iron plays an essential role in oxygen and electron transfer in human body and is also necessary for the synthesis of haemoglobin [32,33]. Adamu et al. [22] figured that the deficiency of iron was the leading causes of anaemia, poor resistance to infection, weakness. Iron is also an essential component of many proteins and enzymes in the human body. The permissible limit of iron set by FAO/WHO (1984) in edible plants was 20 ppm. The highest iron concentration of 0.03 mg/L in Nerium oleander was found in both water and acetone extracts.

5. Conclusions

The present study demonstrated that extraction solvent plays a critical role in the recovery of phenolic and flavonoid compounds from Nerium oleander, Bulbine frutescens, and Cotyledon orbiculata. Among the three species investigated, N. oleander consistently exhibited higher total phenolic and flavonoid contents, indicating a greater abundance of antioxidant phytochemicals. Dichloromethane proved to be the most effective solvent for phenolic extraction, whereas methanol and hexane yielded the highest flavonoid concentrations depending on plant species.

Statistical analysis revealed that solvent type significantly influenced total phenolic extraction, while no significant differences were observed between the phenolic contents of B. frutescens and C. orbiculata. However, the significant interaction between solvent and plant species highlighted that extraction efficiency is species-dependent. Furthermore, the flavonoid profiles of B. frutescens and C. orbiculata differed significantly, as indicated by the Wilcoxon signed-rank test.

ICP-OES analysis showed that N. oleander extracts contain nutritionally important macro-elements, including calcium, magnesium, sodium and potassium, together with trace levels of manganese, zinc and iron. Potentially toxic elements such as lead, chromium and nickel were not detected, suggesting a favourable elemental profile. Methanol extracts contained the highest concentrations of calcium and magnesium, whereas hexane extracts were enriched with potassium.

Overall, the results highlight the importance of solvent selection in maximizing phytochemical recovery and demonstrate that these medicinal plants, particularly N. oleander, represent promising sources of antioxidant compounds and essential mineral nutrients. Further studies should focus on compound isolation, identification of individual phenolic and flavonoid constituents using chromatographic techniques, evaluation of antioxidant and biological activities, and assessment of toxicity and safety to support their development for pharmaceutical, nutraceutical and cosmeceutical applications.