NJ025-1002

Phytochemical Profiling and Kinetic Study of DPPH⦁ Scavenging Activity of Sophora flavescens Aiton Root Extract 

Jongkeun Choi*

Department of Chemical and Biological Engineering, Chungwoon University, Incheon 22100, Republic of Korea.

Corresondence to: Jongkeun Choi, jkchoi@chungwoon.ac.kr

Received: October 11, 2025; Revised: November 24, 2025; Accepted: November 30, 2025; Published: December 2, 2025 

NATPRO J. 2025, 2, 39-45 

https://doi.org/10.23177/NJ025.1002 

Copyright ©  The Asian Society of Natural Products

Abstract

In this study, the antioxidant activity of the traditional medicinal herb Sophora flavescens Aiton root was evaluated through analysis of phytochemicals and DPPH⦁ scavenging activity of 70% ethanol extract. For chemical analysis, ultra performance liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (UPLC-PDA-ESI-MS/MS) was used, and six phytoconstituents, maackiain, formononetin, xanthohumol, kurarinone, lupenone and kuraidin were identified. Of these, five were classified as prenylated flavonoids. The extract, obtained by maceration in 70% ethanol at room temperature for 7 days, showed an extraction yield of 21.3% (w/w, based on dry root weight) and a total polyphenol content (TPC) of 25.4 mg gallic acid equivalents (GAE) per gram of dry root. In the steady-state DPPH⦁ assay, the extract exhibited moderate antioxidant activity with an EC50 value of 674 µg/mL compared with the positive control, gallic acid (EC50 = 2.62 µg/mL). However, in the kinetic study, the S. flavescens extract exhibited a fast initial rate. The sample was rapidly mixed with the DPPH⦁ solution, and absorbance was recorded every 0.5 s for 20 s. The initial reaction rate was then determined from the DPPH⦁ absorbance decay curve. From plots of initial rate data on the log-log scale, the following reaction orders were estimated: 1.05 for the DPPH⦁ and 0.69 for the extract. The pseudo-second-order rate constant (k2) was calculated to be 2.07 mM-0.69 s-1, which is about 19-fold higher than that of gallic acid (0.109 mM-0.48 s-1). These results indicate that the S. flavescens root extract exhibits moderate antioxidant activity at equilibrium, but very high initial radical-scavenging activity. This characteristic antioxidation activity, along with further beneficial results, demonstrates its potential for use as a fast-acting antioxidant ingredient in functional foods and cosmeceuticals, particularly for mitigating acute oxidative stress. 

Keywords

Sophora flavescens; prenylated flavonoids; DPPH⦁ assay; kinetic analysis; antioxidant activity 

Introduction

Sophora flavescens Aiton (called Gosam in Korea), a perennial herb of the Fabaceae family, has roots that have been used for centuries in East Asian traditional medicine for antipyretic, anti-inflammatory, diuretic, and dermatological indications [1]. According to the Dongui Bogam, the Korean medical classic, S. flavescens is cold in nature, bitter in taste, and non-toxic, and it was used to treat skin diseases attributed to 'heat-toxin wind' as well as Hansen’s disease. Phytochemical studies have revealed that S. flavescens contains quinolizidine alkaloids such as matrine and oxymatrine, along with numerous highly bioactive prenylated flavonoids. These constituents have gained attention for their broad pharmacological activities, including anticancer, anti-inflammatory, antimicrobial, and antioxidant effects [2–6].

Oxidative stress induced by reactive oxygen species (ROS) is regarded as a central mediator of aging, inflammatory disorders, and various chronic diseases which has prompted growing interest, in natural antioxidants capable of modulating ROS [7–9]. Although S. flavescens extracts have been reported to possess potent antioxidant activity, most studies have been limited to assessing end-point radical scavenging capacity after a fixed reaction time (e.g., 30 min), [10,11]. However, because oxidative stress in vivo can surge explosively over very short time scales, the initial reaction rate of an antioxidant may be a critical determinant of its biological effectiveness [12–15].

Accordingly, we conducted chemical profiling of a 70% ethanol extract of S. flavescens roots using UPLC-PDA-ESI-MS/MS and evaluated DPPH⦁ scavenging activity using both steady-state and reaction-kinetic analyses. Through this approach, we aimed to provide an in-depth understanding of the antioxidant actions of S. flavescens extract and to clarify its practical potential as a functional material requiring rapid antioxidant responses under acute oxidative stress conditions. 

Materials and methods

Instruments and reagents

S. flavescens Ait. root was supplied by Human Herb Co., Ltd. (Gyeongsan, Korea), which collected, dried, and quality-controlled the material during May–June 2021. The sample was homogenized using a laboratory blender, stored in airtight plastic bags, and used as needed. HPLC grade methanol and acetonitrile (Fisher Scientific, Rockford, IL, USA) and ultrapure water (≥ 18.3 MΩ·cm) were used for extraction of S. flavescens root and preparation of mobile phases of UPLC. For antioxidant assays and total polyphenol determination, 1,1-diphenyl-2-picrylhydrazyl (DPPH, ≥ 95%), gallic acid (≥ 98%), Folin–Ciocalteu phenol reagent, and sodium carbonate (≥ 99.5%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other analytical-grade reagents were purchased from Sigma-Aldrich or Duksan Pure Chemicals (Seoul, Korea). UV–Vis absorbance was measured using an Agilent 8453 UV-Vis spectrophotometer (Agilent Technologies, Waldbronn, Germany) and a SpectraMax Plus 384 microplate reader (Molecular Devices, San Jose, USA). Qualitative analysis of the S. flavescens extract was conducted on a Waters ACQUITY UPLC system equipped with a Micromass Quattro Micro API mass spectrometer (Micromass, Manchester, UK), using a BEH C18 column (2.1 × 100 mm, 1.7 µm; Waters) under a water/acetonitrile gradient containing 0.1% formic acid at a flow rate of 0.3 mL min⁻¹.

Extraction and isolation

Extraction of S. flavescens root was carried out as follows. Dried roots were ground to a uniform fine powder. An accurately weighed 20 g sample was mixed with 200 mL of 70% (v/v) aqueous ethanol (w/v solid-to-solvent ratio of 1:10). After shaking at 25 °C for 7 days, the mixtures were filtered under reduced pressure through Whatman No. 2 filter paper, and if necessary, further clarified using a 0.45 μm PVDF syringe filter prior to further experiments.

Phytochemical profiling by UPLC-PDA-ESI-MS/MS

Profiling of S. flavescens constituents by UPLC-PDA-ESI-MS/MS was performed on an ACQUITY UPLC system (Waters, Milford, MA, USA). Samples were filtered through a 0.22 µm PVDF syringe filter prior to analysis. Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm; Waters). The mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B). A gradient program was used: 10% B from the beginning, linearly increased to 100% B within 40 min, maintained at 100% B for 5 min, and then returned to initial conditions by 46 min for column re-equilibration. The flow rate was 0.30 mL min-1, the column temperature was 35 °C, the injection volume was 3 µL, and PDA spectra were collected from 200 to 500 nm.
Mass spectrometry analysis was performed using a Quattro Micro API mass spectrometer (Waters–Micromass, UK) with UPLC system in electrospray ionization (ESI) mode. Both positive and negative ion modes were used. The capillary, the extractor and the cone voltage were set at 3.5 kV, 3.0 V, and 40 V, respectively. The temperature of the source and desolvation was 120 and 350 °C, respectively. The flow rates of desolvation gas (N₂) and cone gas were 900 L h-1 and 50 L h-1, respectively. Mass spectra were collected in the [m/z 200–1300] interval. All data were collected and processed using MassLynx 3.1 software (Waters), and S. flavescens-derived compounds were putatively identified by a combination of UV absorption spectra, molecular ions, and characteristic fragment ions.

Total polyphenols (TPC)

Total polyphenol content (TPC) was measured by the Folin–Ciocalteu (F–C) method adapted to a 96-well microplate format [16,17]. The S. flavescens extract was serially diluted with distilled water to the calibration range (5–100 μg mL-1 gallic acid equivalent, GAE). Each diluted sample aliquot (70 μL) was dispensed into the well, followed by addition of 70 μL of 1 N Folin & Ciocalteu’s phenol reagent and allowed to stand at 25 °C for 5 min. Subsequently, 70 μL of 7% (w/v) Na2CO3 solution was added, mixed and the mixture was incubated in the dark for 30 min. Absorbance was measured at 760 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices, USA). A blank that had been prepared with distilled water under same conditions was used as a blank. Gallic acid standards (5–100 μg mL-1) were measured in parallel to make a linear calibration curve relating concentration to absorbance. The gallic acid equivalent concentration (GAE, mg mL-1) of samples was converted to mg GAE g-1 dry weight (DW) of extract using the calibration equation. Analyses were performed in quadruplicate, and results were expressed as mean ± standard deviation.

DPPH⦁ scavenging activity

DPPH⦁ scavenging activity was measured by adapting the method of Dietz et al. to a 96-well microplate format [18,19]. The extract was serially two-fold diluted in ethanol to prepare test solutions. To each well of a clear, flat-bottom 96-well plate containing 100 μL of sample, 100 μL of 0.1 mM DPPH⦁ in ethanol was added and rapidly mixed. The plate was incubated protected from light at 25 °C for 30 min, after which absorbance was read at 517 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices, USA). Wells receiving ethanol instead of sample served as the negative control (control), and wells in which both the sample and DPPH⦁ solution were replaced with ethanol served as the blank for baseline correction. DPPH⦁ scavenging activity (%) was calculated as:

where [Asample] is the absorbance of the sample-treated well and [Acontrol] is the absorbance of the negative control well. All experiments were performed in quadruplicate. The resulting scavenging fraction–concentration curves were fitted using non-linear regression in Prism 10.0 (GraphPad Software, USA) to determine EC50 values. Gallic acid was used as a positive control under identical conditions.

Determination of the rate constant for DPPH⦁ scavenging

The reaction kinetics between the 70% ethanol extract of S. flavescens and DPPH⦁ were evaluated by modifying the method of Nikiforov et al. [20]. Briefly, 2 mL of DPPH⦁ solution at various concentrations was transferred to a quartz cuvette with a 1 cm path length, followed by rapid addition of 1 mL of the extract using a micropipette to give a total volume of 3 mL (volume ratio 2:1). Absorbance at 517 nm was recorded continuously at 0.5 s intervals using a Hewlett-Packard 8453 diode-array UV-Vis spectrophotometer equipped with an Agilent 89090A thermostatted cell holder (La Jolla, CA, USA). The reaction mixture was continuously and homogeneously stirred with a small magnetic bar at 200 rpm.

Assuming the overall reaction [DPPH⦁ + AOH → DPPHH + AO⦁], the initial rate v₀ was expressed as v₀ = k2 [DPPH⦁]α [AOH]β [15,21]. By systematically varying the initial concentrations of DPPH⦁ and the extract, initial rates v₀ were obtained and logarithmically transformed to determine the reaction orders α and β from the slopes of the log [v₀]–log [DPPH⦁]₀ and log [v₀]–log [AOH]₀ plots, respectively, and the corresponding rate constant k2 was then calculated. The Beer–Lambert law with ε517 = 11,500 M⁻¹ cm⁻¹ was applied to convert absorbance to DPPH⦁ concentration, enabling quantitative assessment of the radical scavenging kinetics of the S. flavescens extract. 

Results and Discussion

Extraction yield, total polyphenol content, and antioxidant activity

We determined the extraction yield, total polyphenol content (TPC), and DPPH⦁ scavenging activity of S. flavescens root extract prepared with 70% aqueous ethanol and compared these values with those in the literature for other herbal medicinal plants extracted under the same solvent condition and for aqueous extractions (infusions) extracted under comparable conditions (Table 1). In water–ethanol solvent systems, 60–80% ethanol is frequently reported to provide high extraction yields and to effectively recover both hydrophilic and lipophilic bioactive constituents [12,18,21]. Accordingly, we used 70% ethanol, which is consistent with our previous results showing superior antioxidant indices at this level [12,18,21]. 

The extraction yield of S. flavescens was 21.3 ± 1.2%, which is intermediate relative to our prior reports for Lonicera japonica (29.3%) [12] and Chrysanthemum zawadskii (17.3%) [21], and similar to previously reported yields for 70% EtOH extracts of Pueraria lobata (Willd.) Ohwi (21.1%) [22]. It was also slightly lower than values reported for Scutellaria baicalensis (26.91%) and Chrysanthemum indicum (25.47%), and higher than that of Hovenia dulcis (7.26%) [22,23]. Substantial differences in yield under identical solvent conditions are likely attributable to plant-specific factors such as cell wall architecture, tissue density, and intrinsic metabolite composition.

TPC was 25.4 ± 0.9 mg GAE per g of extract which was determined by the Folin–Ciocalteu method (Figure 1). This value is relatively lower than the TPC previously reported for L. japonica extract (57.3 mg GAE/g) [12]. This indicates that the antioxidant activity of S. flavescens may not solely associated with bulk phenolic content but may be dependent by a limited set of constituents with favorable redox-active structures. 

Antioxidant activity of the S. flavescens extract was evaluated using the DPPH⦁ scavenging assay. Based on the concentration response curve (Figure 2), EC50 was calculated as 674 μg mL-1, which was approximately 257-fold higher than that of gallic acid (2.62 μg mL-1). This indicates a moderate overall antioxidant capacity, and it is consistent with previous reports that complex extracts are less potent than single compounds [24,25]. 

Phytochemical profiling with a UPLC-PDA-ESI-MS/MS

To elucidate the chemical composition of the extract, UPLC-PDA-ESI-MS/MS analysis was performed. As shown in Figure 3, several peaks were observed in the PDA chromatogram and in the total ion chromatograms (TIC). By comparing TIC, molecular ion peaks ([M–H]- or [M+H]+), and MS fragmentation patterns with literature data, six constituents were identified: maackiain, formononetin, xanthohumol, kurarinone, lupenone, and kuraidin (Table 2) [26,27]. Notably, most of these are prenylated flavonoids bearing a prenyl substituent on the flavonoid skeleton. Prenylation increases molecular lipophilicity, which can facilitate access to and permeation through phospholipid membranes, potentially enhancing antioxidant action in the membrane microenvironment where oxidative stress predominantly arises [28]. 

Kinetic characteristics of the DPPH⦁ scavenging reaction

To supplement steady-state measurements and investigate the kinetic characteristics of the scavenging reaction, we carried out a time-resolved study. After rapidly mixing the extract with the DPPH⦁ solution, changes in absorbance at 517 nm were recorded every 0.5 s for 20 s. As shown in Figure 4, a steep initial decay was observed, with about 60% of radicals depleted within 5 s, after which the decay proceeded more slowly. This behavior is thought to result from a fast electron-transfer (ET) step followed by slower subsequent processes, as reported for the quercetin–DPPH⦁ system [29]. Using the data from the initial 5 s, initial rates (v₀) were obtained and used to determine empirical reaction orders. The order with respect to DPPH⦁ (α) was 1.05 ± 0.16 and the order with respect to the extract concentration (β) was 0.691 ± 0.067 (Figure 5). Therefore, the reaction is first order with respect to DPPH⦁ but follows a sub-unity partial order with respect to the multicomponent extract, indicating a complex reaction mechanism. It is thought that the rate is likely governed by collisions of DPPH⦁ and more than one component in the extract, and/or by reversible complex formation (such as π-stacking or hydrogen bonding) occurred prior to electron transfer [24,29]. For gallic acid, β was 0.48, also indicating mechanistic complexity. Shojaee et al. [30] reported that even for pure compounds such as gallic acid, k₂ may have values that differ by several orders of magnitude depending on the ability of the solvent to form hydrogen bonds and the polarity. 

Meanwhile, the calculated rate constant, k2 was 2.07 mM-0.69 s-1, which is approximately 19 times higher than that of gallic acid under same conditions (k2 = 0.109 mM-0.47 s-1). Here, k₂ denotes a pseudo-rate constant derived from non-integer reaction orders (α = 1.05, β = 0.69), rather than a strict second-order rate constant in the classical sense (Table 3). This suggests that the S. flavescens extract exhibits very rapid initial reactivity that is not obvious from the EC50 value alone. Indeed, the static DPPH⦁ scavenging assay gave an EC50 of 674 μg mL-1 which is larger than that of gallic acid, 2.62 μg mL-1, meaning lower antioxidant capacity on a dose basis. However, kinetic analysis simultaneously gives reaction rate, reaction orders, and rate constants , which make it possible to evaluate the activity of sample at the initial reaction stage [31,32]. In sum, although overall radical-quenching capacity of S. flavescens extract may be lower than that of gallic acid, the rate of quenching upon encounter is markedly faster. Therefore, S. flavescens extract can be considered a potent, fast-acting antioxidant with high electron-donating kinetics. 

The higher initial reactivity and fractional reaction order observed for the S. flavescens extract could probably be attributed to the distinctive structural features of prenylated flavonoids among its constituents. Prenylated flavonoids, which are flavonoids with an isoprenyl unit, have been reported to exhibit significantly higher antioxidant activity relative to non-prenylated flavonoids [28]. According to Santos and Silva [28], prenylation enhances electron-donating ability and increases radical-scavenging activity through electron-transfer mechanisms with DPPH⦁, possibly due to the increase of the electron density and reactivity of phenolic hydroxyls. It was also reported by Bo et al. [33] that longer prenyl chains substantially increase antioxidant activity, and that both hydrogen-atom transfer (HAT) and single-electron transfer (SET) pathways may involve in parallel. These features account for the observed fractional order for extract (β = 0.691) and biphasic time course. The increased lipophilicity due to prenylation can also improve membrane permeability and this may lead to higher reaction rates at lipid peroxidation sites, thereby affording better protection again oxidative damage. In conclusion, the good DPPH⦁ scavenging rate and the fast kinetics of S. flavescens extract may be attributed to the synergistic effects of its complex multicomponent composition and the enhanced mechanisms of prenylated flavonoids. Therefore, S. flavescens extract can be considered a promising natural, rapid-acting antioxidant, making it a valuable ingredient for the development of anti-aging health foods and cosmetic products. 

Conclusion

The antioxidant activities of a 70% aqueous ethanol extract of S. flavescens root were investigated through steady-state and kinetic studies. UPLC-PDA-MS/MS analysis identified six constituents (maackiain, formononetin, xanthohumol, kurarinone, lupenone, and kuraridin), and its total polyphenol content was determined to be 25.4 mg GAE g-1. In the steady-state DPPH⦁ assay, the extract's EC50 value (674 μg mL-1) was significantly higher than that of gallic acid (2.62 μg mL-1), implying a lower total antioxidant capacity at equilibrium. However, as noted by Kedare and Singh [34], such steady-state assays may not fully capture the dynamics of reactive species in vivo. In contrast, kinetic analysis revealed the extract's rapid action: its initial rate constant (k₂) was 2.07 mM-0.69 s⁻¹, approximately 19 times higher than that of gallic acid, indicating exceptionally fast electron donation. Given that ROS-mediated cellular damage can progress within very short time scales, such fast initial kinetics may be more critical for mitigating oxidative injury than total capacity alone.
This distinctive kinetic profile is likely attributed to the mainly prenylated flavonoids identified in the extract. The prenyl group enhances lipophilicity, which facilitates access to and permeation of phospholipid bilayers—the primary sites of oxidative damage. This allows for faster and more efficient antioxidant intervention. Furthermore, the prenyl groups are known to contribute to radical stabilization [28, 35, 36]. In conclusion, the S. flavescens root extract is characterized not by high total capacity but by its exceptionally rapid radical-quenching reaction kinetics. This rapid-acting profile is particularly advantageous for counteracting acute oxidative bursts, such as those from UV exposure or inflammation. These findings support its potential as a functional ingredient in cosmetics and nutraceuticals designed for immediate antioxidant protection.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

The author declares that there are no competing interests.

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