Trends Sci. 2025; 22(9): 10306

Tithonia diversifolia (locally known in Indonesia by various names such as daun insulin, paitan, rondosemoyo, kembang bulan, kayu paik, kipait and harsaga) is a flowering plant from the Asteraceae family that is known to have a wide range of health benefits [1] and is traditionally used as a herbal remedy in various tropical countries, including Indonesia [2]. This plant contains bioactive compounds such as tannins, saponins, flavonoids, and terpenoids [3], which demonstrate antibacterial, anti-proliferation, anti-inflammatory, analgesic, antioxidant, and antidiabetic activities [4]. However, a detailed identification of these bioactive compounds is crucial to confirm their pharmacological potential. LC-MS/MS is a very sensitive analysis method to identify phytochemical profiles in plant extracts [5]. In this study, LC-MS/MS was used to identify specific compounds in aqueous extract from T. diversifolia leaves.

Bioactive compounds in plant extracts are known to be susceptible to degradation in sub-ideal environmental conditions. This challenge directly affects the effectiveness of lant extracts in their utilization as medicinal plants [6,7]. To overcome this issue, stabilization techniques like nano-emulsions and encapsulation help extend shelf life and maintain compound efficacy. Microencapsulation is a technique that functions to protect core compounds with a coating layer in the form of small particles [8]. Microencapsulation with drying techniques such as spray drying is fast, simple, and feasible to scale [9].

The selection of polymers as coating materials for microcapsules greatly affects the release characteristic, stability, and toxicity of the resulting microcapsules [10]. Chitosan is a polysaccharide obtained from crab and shrimp shell waste. Chitosan is widely used in the microencapsulation process because of its biodegradability, non-toxicity, and high biocompatibility [11]. The structure of chitosan is formed from 2 types of units, namely the acetylated unit (β(1-4)-N-acetyl-D-glucosamine) and the deacetylated unit (D-glucosamine). The process of chitosan deacetylation takes place in 2 stages, using sodium hydroxide and water as auxiliary ingredients [12].

The microcapsules that have been made are further analyzed to determine the ability of antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent which is considered more effective, has a simple, fast, and sensitive method. Antioxidant activity is expressed in IC₅₀ values that can inhibit 50 % of free radicals from DPPH. A low IC₅₀ value indicates higher antioxidant activity [13]. Antioxidants are necessary to combat oxidative stress, therefore, they are able to prevent and repair damage caused by free radicals [14]. The release profile was evaluated at pH 2.2 and 7.4 to simulate physiological conditions in the human body, representing the stomach and intestinal or bloodstream environments, respectively. These pH levels were chosen to assess how effectively the microcapsules protect and release active compounds in conditions mimicking the gastrointestinal tract [15,16].

This study developed a microencapsulation method for a aqueous extract from T. diversifolia leaves using chitosan with spray drying technique. The biological activity of the optimal microcapsule was carried out by conducting an antioxidant activity test. In vitro testing aims to evaluate the effect of microencapsulation on the release and stability of bioactive materials and their interaction with blood sugar regulatory cells. Characterization of microcapsules using SEM and FTIR was carried out to determine the surface morphology of microcapsules and identify the functional groups of microcapsules.

While numerous studies have investigated the bioactive potential of T. diversifolia research on the stability and release mechanism of its active compounds remains limited. Most prior studies have primarily focused on identifying bioactive compounds without further exploring formulations that could enhance their stability and efficacy. Therefore, this study employs the spray drying method to microencapsulation aqueous extract from T. diversifolia leaves, aiming to enhance its stability, prolong shelf life, and regulate the release of active compounds. This approach has been scarcely explored in previous research and holds significant potential for expanding the pharmaceutical applications of aqueous extract from T. diversifolia leaves.

Materials and methods

Chemicals and instrumentation

The research materials used were purchased from Merck: Chitosan (low molecular weight, 50,000 - 190,000 Da), sodium tripolyphosphate (Na-TPP, technical grade, 85 %), quercetin (95 % HPLC, solid), ascorbic acid (pharmaceutical secondary standard), 2-2-diphenyl-1-picrylhydrazyl (DPPH), aluminum chloride (anhydrous, powder, 99.99 % trace metals basis), and phosphate buffered saline (PBS). The leaves of Tithonia diversifolia were collected by UPT Materia Medica in Batu, East Java, Indonesia, and their identity was confirmed through a species determination letter with the voucher number 074/794/102.20-A/2022. The instruments used in this study were Scanning Electron Microscope (SEM with TM 3000 Hitachi), Fourier Transform Infrared Spectrometer (FTIR with type Shimadzu Prestige 21), and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS with Thermo Fisher Scientific).

Extract preparation

The extraction of T. diversifolia leaves was performed using the cold extraction method, specifically maceration with aqueous solvents. The leaves powder was weighed to 100 g and 1,000 mL of aqueous solvent was added in a 1:10 ratio. The maceration process was conducted for 24 h. The aqueous extract was filtered using Whatman No. 1 filter paper (11 cm diameter, ± 11 µm pore size) prior to further analysis. Evaporation of the extract was carried out using a rotary evaporator at 80 - 90 °C and 70 rpm until a thick extract of T. diversifolia leaves was produced. Finally, the extract was stored at 4 °C for further analysis [17].

Phytochemical screening

A confirmatory qualitative analysis of plant extracts was conducted to determine the major classes of compounds, including flavonoids, saponins, alkaloids, tannins, steroids, and terpenoids, using standardized procedures [18-20].

Test for flavonoids

A total of 0.01 g of aqueous extract from T. diversifolia leaves was heated in 10 mL of water. The resulting solution was then filtered. After filtration, 3 drops of HCl and 0.1 g of magnesium ribbon were added. Any changes in color or appearance were then observed.

Test for saponins

A 0.01 g of the aqueous extract from T. diversifolia leaves was taken and mixed with hot water. The mixture was then shaken for 10 s, resulting in the formation of stable foam (1 - 10 cm) for 30 min. Afterward, 1 drop of 2N HCl was added and observations were made.

Test for alkaloids

A total of 0.01 g of the aqueous extract from T. diversifolia leaves was dissolved in 5 mL of chloroform and then filtered to obtain the filtrate. Next, 1 mL of 2 M HCl was added to the filtrate, and the top layer was mixed with the Mayer-Wagner reagent. The formation of a cream or brown-red precipitate indicated the presence of alkaloids. Any changes were then observed.

Test for tannins

A 0.01 g of the aqueous extract from T. diversifolia leaves was placed in a test tube and boiled with 10 mL of distilled water. Then, 1 % ferric chloride (FeCl₃) was added and mixed until homogeneous. The appearance of a blue-black coloration indicated the presence of tannins.

Test for steroids

A total of 0.01 g of the aqueous extract from T. diversifolia leaves was dissolved in 2 mL of chloroform. Then, 3 mL of sulfuric acid (H₂SO₄) was slowly added until distinct layers formed. The appearance of a red top layer and a greenish bottom layer indicated the presence of steroids. Any changes were then observed.

Test for terpenoids

The presence of terpenoids was identified by the formation of a reddish-brown color. In this test, 0.01 g of the extract was dissolved in methanol, followed by the addition of 1 mL of anhydrous acetic acid. Then, 3 mL of sulfuric acid (H₂SO₄) was added. Any resulting changes were observed.

Microencapsulation procedures

The process began with 0.5 g of aqueous extract from T. diversifolia leaves being weighed and then dissolved in 17.5 mL of distilled water. Then, 50 mL of chitosan solution with varying concentrations of 0.05, 0.1 and 0.2 % (w/v) in 2 % (v/v) acetic acid (pH 4) was gradually added while stirring with a magnetic stirrer at 50 rpm for 60 min. Afterward, 200 mL of Na-TPP solution was gradually introduced, and the mixture was stirred for 90 min. The resulting colloidal chitosan microcapsules containing the aqueous extract from T. diversifolia leaves and Na-TPP were then dried using a Buchi spray dryer at an inlet temperature of 105 °C, an outlet temperature of 85 °C, and an air pressure of 1 bar to produce microcapsule powder [21].

The microcapsule preparation was repeated under modified conditions, in which 0.5 g of aqueous extract from T. diversifolia leaves was dissolved in 17.5 mL of distilled water. This time, 50 mL of chitosan solution, at the optimal concentration determined in the previous preparation, was gradually introduced into the pH 4 acetic acid (2 % (v/v) solution while stirring at 500 rpm for 60 min. The mixture was then combined with 200 mL of Na-TPP solution, which was added incrementally, with stirring times varied at 60, 90 and 120 min. The resulting colloidal chitosan microcapsules and Na-TPP encapsulated leaves extract were dried under the same Buchi spray dryer conditions, producing microcapsule powder [21].

Total flavonoid content determination

The total flavonoid content was determined using a colorimetric method [22,23], with quercetin used as the standard to create a calibration curve for flavonoid quantification. A 0.05 g sample of the extract and microcapsules was weighed and dissolved in 5 mL of methanol. The solution was then incubated at 40 °C for 45 min and centrifuged at 1,000 rpm for 10 min. From the supernatant, 0.6 mL was taken and mixed with 0.6 mL of a 2 % aluminium chloride solution. The mixture was incubated at room temperature for 23 min. The absorbance of the resulting solution was measured at 420 nm, corresponding to the maximum wavelength of quercetin. The total flavonoid content was then calculated using the quercetin standard curve and expressed as mg QE/g (milligrams of quercetin equivalent per gram) [21].

Antioxidant activity assay using DPPH

Antioxidant activity was analysed for 3 samples: Microcapsules, extracts, and ascorbic acid. Multiple concentrations were prepared for each: Microcapsules (120, 140, 160, 180 and 200 μg/mL), extracts (20, 40, 60, 80 and 100 μg/mL), and ascorbic acid (2, 4, 6, 8 and 10 μg/mL). From each concentration, 3 mL of solution was transferred into a dark vial, followed by the addition of 2 mL of DPPH (2,2-diphenyl-1-picrylhydrazyl) solution. A control solution was prepared by mixing 3 mL of ethanol with 2 mL of DPPH in a dark vial. All mixtures were homogenized and incubated for 20 min in the dark to minimize light interference with the DPPH method. The absorbance was measured at 516 nm, the maximum wavelength of DPPH. Antioxidant activity was calculated using the formula [24]:

The IC₅₀ value, representing the concentration required to inhibit 50 % of free radical activity, was used to determine the best antioxidant activity. IC₅₀ values were also calculated for comparator antioxidants to assess the potency of the extract compared to proven standards. IC₅₀ was determined using a linear regression equation, with sample concentration on the x-axis and antioxidant activity percentage on the y-axis. By substituting 50 for y, the corresponding x value (IC₅₀) was obtained.

Controlled release assay

The release test for active ingredients was conducted using Simulated Gastric Fluid (SGF) with PBS at pH 2.2 and Simulated Intestinal Fluid (SIF) with PBS at pH 7.4. The pH adjustment was carried out using 0.1 M HCl and 0.1 M NaOH. A total of 0.025 g of microcapsules were weighed and mixed with 5 mL of PBS solution. The mixture was incubated at 37 °C with continuous stirring at 100 rpm, and samples were collected at intervals of 30, 60, 90 and 120 min. After each interval, the mixture was centrifuged for 10 min to separate the supernatant from the precipitate for further analysis. A 1 mL sample of the supernatant was transferred to a test tube, and 1 mL of 2 % AlCl₃ solution (w/v) was added. The mixture was shaken until homogeneous and incubated for 20 min at room temperature. Absorbance was measured using a Thermo Scientific UV-Vis spectrophotometer (Genesys 150, Thermo Fisher Scientific, USA) at a wavelength of 420 nm. Sampling was conducted discontinuously, with a separate beaker used for each time interval. The concentration of active ingredients in the SGF and SIF media was determined using a linear equation derived from the quercetin standard curve created earlier [25-27].

FTIR, SEM and LC-MS/MS analysis

The characterization of microcapsules was performed using FTIR and SEM. Fourier Transform Infrared (FTIR) spectroscopy (Shimadzu-Type IR Prestige-21) was employed to identify functional groups in the extracts, chitosan, Na-TPP, and microcapsules. Samples were first dried and compressed into KBr pellets before measurement within the wavenumber range of 4,000 - 400 cm⁻¹. Scanning Electron Microscopy (SEM) using a Hitachi TM 3000 was utilized to observe the morphology of the microcapsules, with magnifications ranging from 500 to 15,000 . Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Thermo Fisher Scientific) was applied to identify unknown compounds by analyzing their mass-to-charge (m/z) ratios.

Data analysis

All methods were performed in triplicates to ensure the reliability and reproducibility of the results. Statistical analysis was conducted using SPSS software version 26, with data analyzed and presented through descriptive narratives and tables. The microencapsulation results, which involved variations in coating concentration and stirring time, were assessed for normality using the Kolmogorov-Smirnov test, followed by a 1-way analysis of variance (ANOVA) at a 95 % confidence level (α = 0.05) to determine statistical significance. These tests were conducted under the assumptions of normally distributed data and homogeneous variance.

Results and discussion

Phytochemical screening of aqueous extract from T. diversifolia leaves

The aqueous extract from T. diversifolia leaves obtained was subjected to further analysis through phytochemical testing. While modern techniques have advanced the determination of phytochemical content, conventional qualitative tests remain commonly used for initial screening of plant phytochemicals. Table 1 summarizes the phytochemical test results for aqueous extract from T. diversifolia leaves.

Table 1 Phytochemical of Aqueous Extract from T. diversifolia.

Phytochemicals are bioactive compounds found in plants that have been shown to possess therapeutic properties. These compounds are considered to have medicinal value and can be used as natural medicines or drugs due to their potential health benefits [29]. Medicinal plants have been traditionally used to treat various diseases due to the presence of bioactive compounds like alkaloids, flavonoids, phenolics, tannins, saponins, steroids, glycosides, and terpenes. These compounds offer pharmacological effects such as antimicrobial, antioxidant, anti-inflammatory, antidiabetic, and other therapeutic properties, which contribute to the medicinal value of the plant [30]. Based on the phytochemical tests conducted aqueous extract from T. diversifolia leaves., Table 1 shows the presence of active compounds such as flavonoids, saponins, alkaloids, tannins, and steroids. Flavonoids are known for their potent antioxidant and anticancer properties. Saponins help inhibit inflammation, while alkaloids exhibit analgesic, antispasmodic, and antibacterial effects. Tannins possess antimicrobial, anti-inflammatory, antifungal, antiparasitic, and antioxidant properties. Steroids are recognized for their antibacterial activity [31,32].

The LC-MS/MS test was employed on the aqueous extract of T. diversifolia leaves to identify, quantify, and characterize chemical compounds, particularly secondary metabolites and bioactive compounds. This method is highly beneficial due to its ability to detect even trace amounts of compounds with exceptional sensitivity and selectivity.

Figure 1 Chromatogram LC-MS/MS of aqueous extract from T. diversifolia leaves.

Table 2 LC-MS/MS interpretation data of aqueous extract from T. diversifolia leaves.

Figure 1 shows the LC-MS/MS chromatogram of the aqueous extract from T. diversifolia leaves and Table 2 presents the corresponding LC-MS/MS data. In addition to the phytochemical screening, the extract was analyzed using LC-MS/MS, revealing the presence of various bioactive compounds, including phenylalanine, indoleacrylic acid, gilluone B, tagitinin A, thyrotundin, tadeonal, and stigmasterol glucoside. Gilluone B is a terpenoid compound with anti-inflammatory and antioxidant properties, which help reduce inflammation and protect cells from oxidative damage [33]. Tagitinin A, identified in this study, has been shown to regulate blood glucose levels, suggesting its potential in diabetes treatment [34]. Thyrotundin has been reported to exhibit antidiabetic potential by decreasing insulin resistance through the upregulation of PPAR (Peroxisome Proliferator-Activated Receptor Gamma) activity, indicating its role as a promising candidate for the treatment of diabetes mellitus and related metabolic disorders [35]. Polygodial, a sesquiterpene compound, is known for its wide range of therapeutic effects, including antifungal, antibacterial, anti-inflammatory, and anticancer activities [36,37]. Finally, stigmasterol glucoside demonstrates both anti-inflammatory and antimicrobial activities, further supporting its therapeutic potential [38,39].

Microencapsulation of aqueous extract from T. diversifolia

The microencapsulation of aqueous extract from T. diversifolia leaves is carried out using spray drying techniques. Table 3 presents the data on total flavonoid content (TFC) and encapsulation efficiency (% EE) of the resulting microcapsules. The microcapsules are prepared by varying the concentration of the chitosan coating and the stirring duration, with the pH maintained at 4. Among the variations, the microcapsules produced with a chitosan concentration of 0.05 % (w/v) and a stirring time of 90 min at pH 4 demonstrated the highest encapsulation efficiency. As a result, these specific conditions were selected for further analysis.

Table 3 Total flavonoid content and encapsulation efficiency (% EE) of microcapsules containing aqueous extract from T. diversifolia leaves.

In this study, microencapsulation was conducted using chitosan as the coating material and sodium tripolyphosphate (Na-TPP) as the cross-linking agent, employing the spray drying method. Chitosan was selected not only due to its biocompatibility and biodegradability but also because of its inherent antioxidant properties. These antioxidant effects are attributed to the ability of the ammonium ion group (NH₃⁺) in chitosan to react with hydroxyl radicals (OH•), thereby neutralizing oxidative stress [40]. Na-TPP serves as a vital cross-linking agent in the formation of chitosan-based nanoparticles. It facilitates the cross-linking process through ionic interactions, particularly with the positively charged amino groups of chitosan. This ionotropic gelation process leads to the formation of stable and compact nanoparticles, which can encapsulate active ingredients more efficiently. Moreover, chitosan’s ability to enhance the penetration of large molecules across mucosal surfaces makes it especially advantageous for drug and bioactive compound delivery. Although chitosan is insoluble in water and most organic solvents, it becomes soluble in aqueous acetic acid due to the protonation of its glucosamine units. This characteristic is essential for preparing the chitosan solution used in the encapsulation process [29].

As shown in Table 3, microcapsules prepared using a 0.05 % chitosan concentration exhibited the highest flavonoid content (0.378 mg QE/g) and an encapsulation efficiency of 75.36 % ± 0.002. These results suggest that the concentration of chitosan plays a significant role in the encapsulation efficiency. Higher efficiency indicates a more effective encapsulation of active compounds, providing better protection against degradation. At lower chitosan concentrations, the resulting particles tend to have a larger surface area, which may enhance the adsorption and retention of flavonoids on or within the particle matrix. Additionally, the stability of the microcapsules can be attributed to the electrostatic interactions between the protonated amine groups (NH₃⁺) of chitosan and the negatively charged components of the plant extract, further reinforced by cross-link formation with Na-TPP [30]. These interactions contribute to the structural integrity and sustained release capabilities of the microcapsules, which are critical factors in controlled delivery systems.

Microcapsule characterization

The characterization of aqueous extract from T. diversifolia leaves microcapsules was conducted using Fourier Transform Infrared (FTIR) and Scanning Electron Microscope (SEM). FTIR analysis, as shown in Figure 2 and interpreted in Table 4, was performed to identify the functional groups in the microcapsules. Meanwhile, SEM characterization, presented in Figure 3 observed surface morphology of the microcapsules.

Figure 2 FTIR spectra of aqueous extract from T. diversifolia leaves, chitosan, Na-TPP, and microcapsules.

Table 4 FTIR spectra interpretation results.

Figure 3 SEM of aqueous extract from T. diversifolia leaves microcapsules at 10,000× magnification.

Figure 2 presents a comparison of FTIR spectra between the aqueous extract of T. diversifolia leaves, chitosan, Na-TPP, and the resulting microcapsules. As shown in Table 4, the FTIR spectrum of the microcapsules exhibits several prominent absorption peaks. The peak at 3,243.21 cm⁻¹ corresponds to -O-H stretching vibrations, indicative of hydroxyl groups commonly found in flavonoids. A peak at 2,926.59 cm⁻¹ is associated with C-H₃ stretching, typical of alkane compounds, while the band at 1,567.41 cm⁻¹ represents -C=C stretching vibrations, characteristic of aromatic ring structures. Additionally, a peak at 1,401.97 cm⁻¹ indicates the presence of alcohol (-O-H) functional groups, and the peak at 1,098.18 cm⁻¹ corresponds to -P-O-C stretching, suggesting the presence of phosphate groups. The FTIR spectrum of the microcapsules shows strong similarity to that of the extract, indicating the presence of active phytochemical constituents. Simultaneously, the spectrum also reflects the characteristic peaks of the chitosan-Na-TPP matrix used as the encapsulating agent. Notably, the peak at 1,098.18 cm⁻¹ confirms the presence of phosphate groups derived from sodium tripolyphosphate, which acts as a cross-linker during the microencapsulation process. The interaction between phosphate ions from Na-TPP and the protonated amino groups (NH₃⁺) of chitosan confirms the formation of a stable polymeric network, thereby supporting the successful encapsulation of the extract’s bioactive components. These findings demonstrate that the encapsulation process using spray drying was effective in coating and protecting the active compounds [41]. Based on Figure 3, the morphology of the resulting microcapsules appears to be generally spherical with smooth surfaces and varied size distributions. This variation may be attributed to the spray drying process, which is influenced by factors such as temperature and feed viscosity. The smooth and rounded structure suggests that the drying temperature used was optimal, promoting rapid droplet solidification and minimizing surface collapse or shrinkage during particle formation [42].

Microcapsule antioxidant activity test

The biological activity of the microcapsules was assessed by testing for antioxidant activity. The in vitro antioxidant activity was evaluated using the DPPH free radical capture test, which is an effective method for analysing the antioxidant potential of an extract or compound. The results of the test are typically expressed in terms of IC₅₀, which refers to the concentration of the sample solution required to inhibit 50 % of free radical activity.

Table 5 Antioxidant activity test.

Bioactive compounds found in medicinal plants offer substantial health benefits, especially as natural antioxidants capable of neutralizing free radicals and reactive oxygen species (ROS). Antioxidant assays are frequently employed to assess the antidiabetic potential of drug candidates, due to the well-established association between oxidative stress and the progression of diabetes mellitus [43,44]. In this study, antioxidant activity was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. DPPH is an organic compound with an unstable nitrogen radical that produces a deep purple solution, which decolorizes upon reduction by an antioxidant [45]. The results showed that microcapsules produced under optimal conditions had an IC₅₀ value of 139.24 µg/mL, which was higher than that of T. diversifolia leaf extract (70.43 µg/mL) and ascorbic acid (8.41 µg/mL). These findings indicate that the antioxidant activity of the microcapsules is comparatively lower. This difference can be attributed to the variation in bioactive compound concentrations among the 3 samples, as antioxidant activity generally increases with higher levels of flavonoids, phenolics, and other active constituents.

The moderate antioxidant activity of the microcapsules suggests that a higher concentration is required to scavenge 50 % of the DPPH radicals. Despite this, the microcapsules still exhibit antioxidant potential, supporting their candidacy for therapeutic applications [27]. One contributing factor to the reduced activity is the confinement of bioactive compounds within the microcapsules, which limits their immediate interaction with DPPH radicals [46]. It is important to note that the primary role of the coating material is not to enhance the antioxidant activity of the core substance but rather to shield it from environmental factors [26].

Moreover, the higher IC₅₀ value of the microcapsules compared to the crude extract can be explained by 2 main factors: (1) Reduced bioavailability of active compounds due to the encapsulating matrix, which can hinder solubility and diffusion, and (2) limited interaction between antioxidants and DPPH radicals as the chitosan coating forms a physical barrier. Since microcapsules release their contents gradually, antioxidant compounds may not be present in sufficient concentration at the time of measurement. To better understand these observations, future studies are recommended to investigate the release kinetics of the encapsulated compounds during antioxidant testing, as well as to compare the concentration of flavonoids and phenolic compounds before and after encapsulation [21,25]. Such insights will be valuable in optimizing encapsulation techniques to balance both protection and bioactivity of plant-derived compounds.

Controlled release of microcapsule

In vitro release tests were conducted on microcapsules at pH 2.2 and pH 7.4, with release durations of 30, 60, 90 and 120 min. These variations in pH and release time were applied to evaluate their impact on the release of the microcapsules. Figure 4 presents the microcapsule release profile, while Table 6 summarizes the percentage of microcapsule release at both pH 2.2 and pH 7.4.

Figure 4 Release profile of bioactive from microcapsule at pH 2.2 and pH 7.4. Different letter notations indicate significant difference between treatments at a significance of α = 0.05.

Table 6 Release profile of bioactive from microcapsule at pH 2.2 and pH 7.4.