Abstract Aging is a natural biological process characterized by the progressive decline of cellular functions, leading to skin alterations such as wrinkle formation and reduced regenerative capacity. Key contributing factors include oxidative stress and the loss of collagen. This study investigates the effects of Coix lacryma-jobi L. (adlay) extracts, which are rich in antioxidant compounds, on human fibroblast (HFF-1) cells. Two types of crude extracts derived from raw (CLR) and cooked (CLC) adlay seeds were prepared using 95% ethanol via ultrasonic-assisted extraction. High-performance liquid chromatography (HPLC) and Ultra-high performance liquid chromatography-Mass spectrometry (UHPLC-MS) were employed to quantify, and identify bioactive compounds, respectively. Cell viability was assessed using the MTT assay, demonstrating that adlay extracts exhibited no cytotoxicity, even at concentrations up to 1,600 µg/mL. The CLC extract significantly reduced intracellular reactive oxygen species (ROS) levels. In addition, a combination of L-ascorbic acid and adlay extracts (50 µg/mL) protected cells against H₂O₂-induced oxidative stress. Co-treatment with CLC and L-ascorbic acid enhanced collagen production in fibroblast cells. These findings suggest that cooked adlay extract has potential for further investigation as a natural source of bioactive compounds for applications related to oxidative stress mitigation and collagen synthesis.

Keywords: Adlay crude extract, Active compound, Antioxidant, Oxidative stress,  Collagen synthesis

Funding: This project was supported by the National Science Research and Innovation Fund (NSRF) of Thailand (Grant NO. R2567B033), Thailand. This work was partially supported by Naresuan University (Grant No. R2562B085 and R2566C051 to MS), Thailand.

Citation: Theangyoo, W., Parhira, S., and Srikummool, M. 2026. Adlay (Coix lacryma-jobi L.) extracts for protecting oxidative stress and enhancing collagen synthesis in skin fibroblast cells (HFF-1). Natural and Life Sciences Communications. 25(4): e2026084.

Graphical Abstract:

INTRODUCTION

Skin aging is a natural process that gradually declines, producing wrinkles. Skin aging is caused by many factors, including ultraviolet (UV) damage, environmental pollution, cellular senescence, oxidative stress, and collagen loss (Zhang and Duan, 2018; Papaccio et al., 2022). The most common factors of aging are the loss of collagen and oxidative stress. Fibroblast cells have an important function on the skin, such as being the extracellular matrix for organs, wound healing processing, and synthesizing collagen fibers (Shoulders and Raines, 2009). In a previous study, fibroblast cells and collagen formation were found to form in older people at a slower proliferation rate than in teenagers (Varani et al., 2006).

Reactive Oxygen Species (ROS), particularly free radicals, are highly reactive molecules that, when present in excess, disrupt cellular redox balance and induce oxidative stress. This imbalance can lead to cellular damage, protein degradation, and the development of age-related diseases (Snezhkina et al., 2019). In skin cells, elevated ROS levels have been shown to inhibit collagen synthesis, partly through the suppression of collagen-related genes such as COL1A2 gene. For example, hydrogen peroxide (H₂O₂), a common ROS, can impair collagen production in fibroblasts, whereas antioxidant treatment has been shown to restore both COL1A2 expression and collagen synthesis. (Palungwachira et al., 2019).

Coix lacryma-jobi L., recognized in the World Flora Online as Coix lacryma-jobi var lacryma-jobi, commonly known as adlay, belongs to the Poaceae family (World Flora Online, 2022). It is a traditional Chinese medicine (Zhu, 2017), with various studies reporting its antioxidant, anti-cancer (Parhira et al., 2025), anti-tyrosinase (Lin et al., 2022), immunomodulatory (Suzuki and Konaya, 2021) and anti-inflammatory (Gharegezloo et al., 2024) properties. Adlay extracts contain phenolic compounds and flavonoids (Kuo et al., 2012; Tan et al., 2017; Zhu, 2017), including substantial amounts of coixol, an important active compound in this plant (Zhu, 2017). Several treatment of adlay seeds before extraction were introduced (Bunthawong and Jomduang, 2016; Jomduang and Budthawong, 2019). However, the effects of the cooked adlay extract on fibroblast cells remain unclear. Therefore, the focus of this study is to investigate and compare the antioxidant effects and collagen synthesis of raw and cooked adlay extracts in fibroblast cells. Here, raw and cooked adlay seed extracts were prepared using ultrasonic-assisted solvent extraction, a cold-process method, providing evidence of coixol's existence and content using UHPLC-MS and HPLC, respectively. The biological activities of the extracts were evaluated in terms of their protective effects against oxidative stress and their ability to promoting collagen production.

MATERIAL AND METHODS

Plant material

The Coix lacryma-jobi L. or Coix lacryma-jobi var. lacryma-jobi seeds (5 kg) were purchased on May 9, 2020, from Raitip company (Thanya Farm Co., Ltd., Nonthaburi, Thailand) and authenticated by a taxonomist before being deposited for reference at the PNU Herbarium, Department of Biology, Faculty of Science, Naresuan University, Phitsanulok (Voucher specimen No. 005348) as described in our previous report (Parhira et al., 2025). The purchasing of the plants from commercial products available in the market and their use for research purposes followed the Thailand national guideline as approval document number 0284 by the Department of Agriculture, Ministry of Agricultural and Cooperatives, Thailand, according to Plant Varieties Protection Act B.E. 2542 (1999) Section 53. The raw seed was blended to prepare a dried powder. The cooked seed powder was prepared by placing 1 kg of raw seed and 1,500 mL of water in a rice cooker (Phillips, Thailand) and steaming at approximately 100°C for 45 min. The cooked seeds were dried in a hot air oven at 50°C, and the powder was produced using a blender. The dry powder from raw and cooked seeds of C. lacryma was stored in airtight containers at room temperature (27 ± 5 °C) until extraction.

Sample preparation

The dry powder from the raw (2 kg) and cooked seed (2 kg) of C. lacryma-jobi was extracted using 95% ethanol [5 L, (Commercial grade, Liquor Distillery Organization, Chachoengsao, Thailand)] and then placed into an ultrasonic bath (GT^® Sonic, China) at room temperature (27 ± 5 °C) for 30 min. The alcoholic filtrate was separated from the plant residue and evaporated using a rotary evaporator (Buchi, Switzerland) at 45°C to obtain the ethanolic extracts of raw (CLR) and cooked (CLC) seeds of C. lacryma-jobi. The percentage yield of the extract was calculated by comparing it to 100 g of the C. lacryma-jobi dried powder (Srisawang et al., 2024).

Determination of coixol content by high-performance liquid chromatography

The coixol content of CLR and CLC was determined by following the suggested protocols (Zhang et al., 2010) with slight modifications. In brief, the CLR or CLC (5 mg/mL in methanol, 20 µL) was injected into the HPLC system (Shimadzu pump LC-10ATvp, Japan) using a C18 column (ACE^® Excel 5 C18, 4.6 mm × 250 mm, 5µm), mobile phase of acetonitrile (HPLC grade, LabScan, Thailand) and 0.1% Phosphoric acid (AR grade, Sigma Aldrich, USA) in water (25:75% v/v) with a flow rate of 0.8 mL/min for 20 minutes, detection wavelength of 230 nm and column temperature of 25°C. The content of coixol (98.78% purity, MedChemExpress, USA) in CLR and CLC was calculated using the coixol standard curve (0.01−0.75 µg/mL, Y = 87696X − 309, R² = 0.9954, where Y represented the peak area at the retention time of 13.466 ± 0.004 min and X the concentration of coixol (µg/mL)). The results were expressed as µg of coixol per one gram of extract (mean ± S.D.) from three independent experiments.

Identification of coixol by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)

The UHPLC-MS was used to identify the existence of coixol in CLR and CLC following the previously reported protocol (Liu et al., 2024). Briefly, the liquid chromatography was performed on an Agilent 1290 UHPLC system coupled with a Waters ACQUITY UPLC^® BEH C18 column (1.7 μm, 2.1 mm × 100 mm). The injection volume of coixol standard solution in methanol (5.0 mg/mL), CLR (10.0 mg/mL), and CLC (10 mg/mL) was 1 µL. Mobile phase consisted of water containing 0.1% formic acid (Phase A), and acetonitrile containing 0.1% formic acid (Phase B). A gradient elution system was performed to separate the compounds in CLR and CLC for 15 min under the following conditions: at 0-8 min, 10%-55% B; 8-11 min, 55%-95% B; 11-15 min, 95% B. Flow rate at 0.35 mL/min, and the column temperature at 40°C were set up. Mass spectra were collected using an Agilent 6545 accurate-mass Q-TOF/MS system. The instrument was operated in positive ion mode. Mass spectra were recorded in the range of 100-1,700 m/z. The capillary and the nozzle voltages were 3,500 V, and 500 V, respectively. The dry gas flow rate was set at 10 L/min, while the dry gas temperature was set at 300°C, the nebulizer pressure at 35 psi, the sheath gas flow rate at 10 L/min, and the sheath gas temperature at 300°C. The extracted ion chromatograms (EICs) at m/z 166.0 for the coixol standard, CLR, and CLC were analyzed using Agilent MassHunter software. 

Determination of phytochemical contents

The total content of selected phytochemicals was determined using colorimetric assays for flavonoids (Pakal and Pyrzynska, 2014; Srisawang et al., 2024), phenolics (Baba and Malik, 2015; Srisawang et al., 2024), and triterpenoids (Wei et al., 2015; Srisawang et al., 2024), following previously reported methods. All experiments were done in triplicate. Details of each protocol are provided in Supplementary Material 1.

Cell culture

The HFF-1 cell line or Human foreskin fibroblasts were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cell is capable of producing extracellular matrix, including collagen. It serves as a model cell for understanding collagen production and synthesis mechanisms, making it well-suited for the objectives of this study. The HFF-1 cells were maintained in DMEM (Gibco, UK) supplemented with 15% FBS (Gibco, UK), and 1% Antibiotics (Gibco, UK). The cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. This project was approved by Naresuan University Institute Review Board No. P10183/63.

Extract treatments

First, the adlay extracts were dissolved with 100% DMSO (Gibco, UK) and then diluted using 0.1% DMSO, which is a non-toxic concentration of DMSO for HFF-1 cells. Additionally, L-ascorbic acid was freshly prepared in culture media with 0.1% DMSO, and the control group was prepared with an equivalent amount of 0.1% DMSO. All samples were prepared by incubation.

Cell viability assay

Cell viability was determined by an MTT assay. The HFF-1 cells were seeded in 96 well plates at 10,000 cells/well, then cultured for 24 hr at 37°C, 5% CO₂ prior to treatment with the extract. The extract was dissolved by 0.1% DMSO. The cells were then treated with various concentrations of adlay extract (25, 50, 100, 200, 400, 800, and 1,600 µg/mL), incubated for 48 hr, and subsequently for another 4 hr with 1 mg/mL MTT (BioBasic, UK) solution. The MTT solution was removed, and the formazan dissolved with DMSO (100 µL/well) and measured at 570 nm using the microplate reader.

Exposure of H₂O₂

H₂O₂ is an inducing agent for oxidative stress. A solution was freshly prepared in PBS. To examine for protection against cell damage, the cells were exposed to H₂O₂ at 100 µM for 1 hr. A solution was freshly prepared in cultured media for the ROS level; the cells were exposed to H₂O₂ at 100 µM for 12 hr. For the control group, the cultured media was replaced with PBS or fresh cultured media.

Cell damage protection by MTT assay

HFF-1 cells were seeded in 96-well plates at 10,000 cells/well and then cultured for 24 hr at 37°C, 5% CO₂ prior to treatment with the extract. The extract was dissolved using 0.1% DMSO. Cells were treated with the extract at concentrations of 50 or 400 µg/mL in separate experimental groups. Additional treatments included 200 µM L-ascorbic acid, and a mixed condition: 50 µg/mL of the extract combined with 200 µM L-ascorbic acid. Control cells received vehicle only. After 48 hr of incubation, cells were treated with 100 µM H₂O₂ in PBS for 1 hr. After removing the supernatant, the wells were washed with PBS and the cells were then incubated with 1 mg/mL MTT solution. After 4 hr of incubation, the MTT solution was removed, and the formazan dissolved with DMSO (100 µL/well). The absorbance of the solution was measured at 570 nm.

ROS level by DCFH-DA assay

The HFF-1 cells were seeded in 24 well plates at 75,000 cells/well, and cultured for 24 hr at 37°C, 5% CO₂ prior to treatment with the extract. The cells were treated with one of the following: 50 or 400 µg/mL of the extract, 200 µM L-ascorbic acid, and the pre-prepared control solution. All the cells were then incubated for 48 hr at 37°C. The cells were then treated with 100 µM H₂O₂ in cultured media for 12 hr. The supernatant was then removed, and the cells were washed twice with PBS. A 10 µM volume of DCFH-DA (Sigma, UK) solution was dissolved in serum-free media, and the solution was treated for 30 min. The supernatant was then removed and the remaining cells washed twice with PBS. The fluorescent signals were measured at 485/535 nm.

Collagen content

The collagen content was detected by the Sirius Red/Fast Green Collagen Staining Kit (Chondrex, USA). The cells were seeded in 24 well plates at 75,000 cells/well, and cultured for 24 hr at 37°C, 5% CO₂ prior to treatment with the extract. The cells were treated with 50 and 400 µg/mL of the extract, 200 and 1,000 µM L-ascorbic acid, mixed condition: 50 µg/mL of the extract combined with 200 µM L-ascorbic acid or control group, and incubated for 72 hr. Next, the supernatant was removed and the wells washed with PBS. Kahle fixative was applied to the cells for 10 min, and the wells were washed with PBS. The dye solution was added to the wells which were then incubated at room temperature (about 25°C) for 30 min and the stain was rinsed with distilled water. The stain on the cells was eluted with the dye extraction buffer. The absorbance of the solution was measured at 540 nm and 605 nm. The collagen content was calculated by OD 540 value OD 605*0.291/0.0378.

Statistical analysis

Student’s t-test was used to compare the two groups to judge the statistical significance. For multiple group comparison, one-way analysis of variance (ANOVA) was used followed by post hoc analysis by LSD. Statistically significant values were set at P < 0.05.

RESULTS

Coixol and phytochemical contents in the extracts

The dry powder of raw (2 kg) and cooked seeds (2 kg) of C. lacryma-jobi were extracted with 95% ethanol (5 L) to obtain the crude ethanolic extracts of raw (CLR, 175.68 g, 8.78%) and cooked seed (CLC, 76.41 g, 3.82%), respectively. The coixol content of CLR and CLC was determined by HPLC. The HPLC chromatograms of blank, coixol standard (0.25 µg/mL), CLR, and CLC are illustrated in Figure 1A–D, respectively. The content of coixol in CLR (2.04 ± 0.08 µg/ g extract) was approximately three times lower than that of CLC (6.51 ± 0.22 µg/g extract), as shown in Figure 2. The existence of coixol (Figure 3A, C₈H₇NO₃, calculated m/z of [M+H]⁺ = 166.0504) in CLR (Figure 3B, found m/z of [M+H]⁺ = 166.0494) and CLC (Figure 3C, found m/z of [M+H]⁺ = 166.0596) was further confirmed by the extract ion chromatograms at m/z 166.0 [M+H]⁺ from UHPLC-MS determination at the peaks at retention time around 3.8 min. The peak height of CLC at 10.0 mg/mL was approximately three times more than that of CLR at the same concentration, in same trend of the HPLC quantitative results.

CLR and CLC contained various phytochemicals, as shown in Table 1. It was found that one gram of CLR and CLC consisted of 1) total flavonoid contents of 5.96 ± 0.30 and 30.89 ± 1.62 mg rutin equivalent; 2) total phenolic contents of 5.38 ± 0.37 and 6.83 ± 0.22 mg gallic acid equivalent; and 3) total triterpenoid contents of 146.53 ± 9.66 and 231.62 ± 4.14 mg ursolic acid equivalent, respectively.

Table 1. Phytochemical contents of the raw (CLR) and cooked (CLC) adlay extracts.

Figure 1. High-performance liquid chromatographic chromatograms of the blank (A), standard coixol 0.25 µg/mL (B), CLR 5mg/mL (C), and CLC 5 mg/mL.

Figure 2. Coixol content (µg/ g extract) of CLR and CLC.

Figure 3. Extracted ion chromatogram (EIC, 166.0, positive mode) of the standard coixol 5 mg/mL (A), CLR 10 mg/mL (B), and CLC 10 mg/mL (C).

Viability of HFF-1 cells

Cells were treated for 48 hr with various concentrations of adlay extract between 0–1,600 µg/mL. Cell viability was determined by MTT assay. The results showed that the concentration of CLR and CLC at 25, 50, 100, 200, 400, and 800 µg/mL had no significant cytotoxic effect (Figure 4a). Hence, concentrations of CLR and CLC at 50 and 400 µg/mL were chosen for subsequent experiments. However, it is important to determine if the higher concentrations have cytotoxic effects on the cell. Therefore, we conducted tests at concentrations of 400, 800, 1,200, and 1,600 µg/mL. Similar to CLR and CLC at these concentrations, the effects were not significantly cytotoxic (Figure 4b).  

Figure 4. Percentage of fibroblast cell viability after being treated with Coix lacryma-jobi L. extracts at (A) concentrations of Coix lacryma-jobi L. extracts at 25, 50, 100, 200, 400, and 800 µg/mL and (B) concentrations of Coix lacryma-jobi L. extracts at 400, 800, 1,200, and 1,600 µg/mL (n = 3; mean ± SD).

Protective effect of extracts from oxidative damage by H₂O₂

To determine the viability of HFF-1 cells exposed to H₂O₂-induced oxidative stress, they were pre-treated with the extracts for 48 hr and 100 µM H₂O₂ for 1 hr. The cytotoxicity was evaluated using an MTT assay. The findings revealed that cell viability decreased to 87% of the control in cells treated with 100 µM H₂O₂ for 1 hr. Pre-treatment with 50 and 400 µg/mL CLR and CLC showed no significant protection against cell damage. However, the group of mixed extracts: M-CLR (50 µg/ml CLR combined with 200 µM L-ascorbic acid), and M-CLC (50 µg/mL CLC combined with 200 µM L-ascorbic acid) protected the cells significantly from oxidative stress by 100 µM H₂O₂ (Figure 5). Therefore, CLR and CLC were considered appropriate for use as a supplement to L-ascorbic acid.

Figure 5. Effects of adlay extracts in preventing H₂O₂-induced cytotoxicity in the fibroblast cells (n = 3; mean ± SD; where NS refers to a non-significant difference between the two groups, # P <0.05 vs H₂O₂; * P <0.05 vs vehicle control).

Intracellular ROS level on HFF-1 cells

DCFH-DA fluorescence was detected when the ROS oxidize the H molecules in the DCFH structure. In this study, the DCF fluorescence intensity was determined after pre-treatment for 48 hr and induced oxidative stress by 100 µM H₂O₂ for 12 hr. The increase in DCF fluorescent intensity was significantly greater in the cells exposed to H₂O₂ than in the non-exposed group. However, our results did not show any decrease in intracellular ROS levels by the cells in the medium and 0.1% DMSO conditions but pre-treatment with the cooked adlay extracts (CLC) did decrease the intracellular ROS levels significantly (Figure 6).

Figure 6. Percentage of DCF positive after pre-treatment with the adlay extracts and exposure to H₂O₂-induced oxidative stress in the fibroblast cells. (n = 3; mean ± SD; * P <0.05 vs vehicle control). 

Collagen content
The Sirius Red/Fast Green Collagen Staining Kit is used to stain the collagen on cells. Sirius Red specifically binds the (Gly-X-Y)_n helical structure, while Fast Green binds to non-collagenous proteins. In this experiment, the collagen was investigated after treatment with the extracts for 72 hr. The collagen staining on cells is shown in Figure 7. The results showed that CLR and CLC at low dose (50 µg /ml) did not significantly increase intracellular collagen but at high dose (400 µg/ml) and M-CLC increased the collagen levels, significantly (Figure 8) in a similar manner to 1,000 µM L-ascorbic acid.

Figure 7. Collagen staining on fibroblast cells. (A) control, (B) vehicle control, (C) CLR 50 µg/mL, (D) CLR 400 µg/mL, (E) CLC 50 µg/mL, (F) CLC 400 µg/mL, (G)  L-ascorbic acid 200 µM, (H) L-ascorbic acid 1,000 µM, (I) CLR 50 µg/mL + L-ascorbic acid 200 µM, (J) CLC 50 µg/mL + L-ascorbic acid 200 µM.

Figure 8. Collagen content in fibroblast cells after treatment with the adlay extracts for 72 hr (n = 3; mean ± SD; a and b show significant differences using one-way ANOVA).

DISCUSSION

Adlay (C. lacryma-jobi) is a traditional Chinese medicinal plant reported to have antioxidant, anti-inflammatory, anti-cancer, and anti-obesity properties. Adlay extracts have also been reported to contain coixol, phenolic compounds, and flavonoids (Kuo et al., 2012; Tan et al., 2017; Zhu, 2017; Parhira et al., 2025). In the present study, the bioactive effects of raw and cooked adlay (C. lacryma-jobi) crude extracts containing different levels of coixol and other phytochemicals were investigated. The results demonstrated that the cooked adlay extract exhibited enhanced protective effects against oxidative stress and showed greater potential to stimulate collagen production in fibroblast cells compared with the raw extract. To our knowledge, this report simultaneously compares the phytochemical profiles and antioxidant activities and is the first to report collagen-promoting effects of raw and cooked adlay extracts. These findings provide new insights into the influence of thermal processing on the biological activities of adlay and highlight the potential of cooked adlay as a valuable source of bioactive compounds for applications in the food, cosmetic, and pharmaceutical industries.

The total contents of the selected phytochemicals were determined according to the previous reports of determination of flavonoids (Pakal and Pyrzynska, 2014), phenolics (Baba and Malik, 2015), and triterpenoid contents (Wei et al., 2015). The cooked crude extract (CLC) was found to contain more coixol (3 times), flavonoids (5.18 times), phenolics (1.27 times), and triterpenoids (1.58 times) than those of the raw crude extract (CLR). This suggests that high-temperature cooking enhances the bioactive content of coix seeds, likely due to a previous report of roasting-induced phenolic production in sesame via the Maillard reaction and the thermal breakdown of the seed matrix (Rizki et al., 2015).

The experimental results shown in Figures 4a and 4b indicate that both crude extracts at concentrations of 25–1,600 µg/mL had no cytotoxic effect. Specifically, in Figure 4a, we observed no cytotoxicity at concentrations of 400 and 800 µg/mL. We also test the higher concentrations of the crude extract, up to 1,600 µg/mL, to determine their impact on cell viability. Subsequently, we aim to minimize the concentration of the extract used in the other experiments. Therefore, two concentrations of adlay extracts at 50 µg/mL and 400 µg/mL were selected to represent low and high treatment levels for further investigation. The higher concentration (400 µg/mL) was chosen based on its solubility and lack of cytotoxicity in the culture system, while the lower concentration (50 µg/mL) was evaluated biological activity at a minimal effective dose. Intermediate doses were excluded to allow a clearer comparison between low and high exposure conditions. Additionally, we anticipated cost savings with significant benefits.

Oxidative stress is an imbalance between free radicals and antioxidants in cells. It can damage cells and tissues, promote cancer, decrease collagen synthesis, and induce cell senescence (Li et al., 2013; Pizzino et al., 2017; Yakaew et al., 2019). The most common factors of skin aging are collagen loss and oxidative stress (Palungwachira et al., 2019). Hence, compounds that can prevent oxidative damage in fibroblasts could be used as skin anti-aging agents. The biological activities of Coix lacryma-jobi L. extracts against oxidative stress induced by H₂O₂ were investigated in this study. To determine the minimum concentration required to induce oxidative stress in cells while maintaining a high percentage of cell viability, we conducted cytotoxicity tests with H₂O₂, and the results indicated that 100 µM H₂O₂ met our criteria (Supplement 2, Table S8). The cell viability data after pre-treatment for 48 hr by MTT assay showed that CLR or CLC non-significantly inhibited cytotoxicity, but the combination of 200 µM L-ascorbic acid and 50 µg/mL of raw (M-CLR), or cooked (M-CLC) adlay extracts, significantly decreased the cytotoxicity of H₂O₂ on HFF-1 cells (Figure 5). These results indicate that the extracts at a low, non-cytotoxic dose in combination with the antioxidant ascorbic acid, could enhance protective effects while reducing the required concentration of the extract. Crude extracts from various plants often contain antioxidant compounds, such as anthocyanins from rice extract (Palungwachira et al., 2019) and walnut extract (Rusu et al., 2020), as well as from fermented adlay extract (Fang et al., 2023), which significantly inhibit H₂O₂-induced cytotoxicity. Although direct pathway validation was not performed in the present study, the observed biological effects are consistent with mechanisms previously reported for adlay-derived phytochemicals. Adlay seed extracts contain various active compounds, phenolic compounds and flavonoids, these may contribute to the observed cytoprotective effects through both direct and indirect antioxidant mechanisms. In addition to scavenging ROS, adlay phenolics have been shown to suppress lipid peroxidation and enhance endogenous antioxidant defenses by increasing super oxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities. Ferulic acid-containing phenolic fractions from adlay activate NRF2 signaling, thereby promoting cellular antioxidant responses and protecting against H₂O₂-induced oxidative damage. Coixol may also contribute to the antioxidant activity of adlay extracts through modulation of redox-sensitive inflammatory pathways. Previous study demonstrated that coixol suppresses NF-κB activation, MAPK signaling, and NLRP3 inflammasome activity. Since ROS-mediated activation of NF-κB and MAPK pathways plays a central role in oxidative stress-induced cellular injury, inhibiting these pathways by coixol may help attenuate oxidative damage and inflammatory responses (Zhao et al., 2017; Hu et al., 2020; Weng et al., 2022; Zeng et al., 2022). 

Furthermore, we observed that the combination of L-ascorbic acid and the extract reduced the cytotoxicity of H₂O₂ on fibroblasts, whereas L-ascorbic acid alone at the same concentration did not significantly inhibit cell cytotoxicity. L-ascorbic acid is one kind of antioxidant molecule which, in combination with CLR or CLC, can be a good supplement for improving the uptake of L-ascorbic acid in the body. CLC significantly decreases the ROS levels (Figure 6), which explains why the L-ascorbic acid-CLC combination at these concentrations may be effective against ROS in cells. The concentration of ascorbic acid (200 µM) was selected based on previously published studies in cell-based models (Yin et al., 2018; Taroncher et al., 2023). This concentration has been used and is considered non-cytotoxic. CLC, also, has higher triterpenoids and flavonoids than CLR, so this compound could be a good extract for reducing ROS in cells. 

Collagen, produced in the fibroblast cells, is the most abundant protein in the body but collagen synthesis is controlled by many factors (Li and Wu, 2018; Matsuda et al., 2006). One factor that affects collagen formation is the presence of antioxidants, which can increase the expression of collagen and collagen proteins (Palungwachira et al., 2019). This study demonstrates that adlay extracts do not affect the promotion of collagen formation, but cells treated with M-CLC (200 µM Vitamin C and 50 µg/ml of cooked adlay extract) do increase collagen formation (Figure 8). Cells demonstrate the process of transcribing mRNA into collagen polypeptide strands, modified to form a triple helical structure. There is an important cofactor associated with this process, namely Vitamin C (Pullar et al., 2017; Li and Wu, 2018), a popular food supplement that helps to strengthen and stabilize the collagen fibrils (Pullar et al., 2017). However, while Vitamin C is a cofactor in the process, too much Vitamin C uptake has toxic side effects such as creating high levels of oxalate in the kidneys, leading to the risk of kidney stones (Assimos, 2004; Ferraro et al., 2016). A variety of natural antioxidants have been studied for their collagen-enhancing effect, such as anthocyanin extraction from Oryza Sativa L., which reportedly has the ability to promote the production of collagen in Primary Dermal Fibroblasts (Palungwachira et al., 2019) while morning glory leaf extract can promote COL1A1 gene expression in CCD-986sk cells (Panichakul et al., 2022).

The greater protective effect against oxidative stress and collagen-promoting activity observed in the cooked adlay extract (CLC) may be associated with its higher levels of coixol, flavonoids, phenolics, and triterpenoids compared with the raw extract (CLR). Consistent with this observation, CLC significantly increased intracellular collagen levels in a concentration-dependent manner, with the highest concentration (400 µg/mL) producing an effect comparable to that of 1,000 µM L-ascorbic acid, whereas CLR showed no significant enhancement (Figure 8). The superior biological activity of CLC may be explained by the increased availability of bioactive phytochemicals following thermal processing. Because excessive ROS activates these signaling cascades, resulting in oxidative stress, inflammation, extracellular matrix degradation, and impaired collagen homeostasis, reducing intracellular ROS may preserve fibroblast function and create a favorable environment for collagen biosynthesis. In addition, natural polyphenols have been shown to promote collagen synthesis through activation of the TGF-β/Smad signaling pathway while simultaneously suppressing collagen degradation via inhibition of matrix metalloproteinases (MMPs), particularly MMP-1 (Żynda et al., 2025). Fang et al. (2023) further demonstrated that coix-derived bioactive compounds suppress MMP-1 expression, suggesting that the higher coixol content in CLC may also contribute to preserving collagen homeostasis. Therefore, the enhanced intracellular collagen accumulation observed in CLC-treated fibroblasts is likely attributable to the combined effects of antioxidant protection, stimulation of collagen biosynthesis, and reduced collagen degradation. Although these molecular mechanisms were not directly investigated in the present study, our findings are consistent with previously reported mechanisms of polyphenol- and coix-derived bioactive compounds and suggest that thermal processing enhances the functional properties of adlay by increasing the availability of bioactive phytochemicals with antioxidant and collagen-promoting activities.

This study has limitations that should be considered when interpreting the findings. First, although coixol was quantified using HPLC and its identity was further confirmed by UHPLC-MS, the chromatographic peak corresponding to coixol was not fully resolved. Consequently, complete analytical validation, including peak purity assessment, specificity evaluation, and robustness testing, was not performed, which may reduce confidence in the quantitative determination of coixol. Therefore, the quantitative coixol data should be interpreted with caution. Future studies should employ optimized chromatographic conditions and fully validated analytical methods to improve the accuracy and reliability of coixol quantification. Second, the biological activities observed in this study were evaluated using crude extracts containing multiple phytochemical constituents. Therefore, direct causal relationships between individual phytochemicals, including coixol, flavonoids, phenolics, and triterpenoids, and the observed antioxidant and collagen-promoting activities could not be established. Further studies using isolated compounds together with gene and protein expression analyses are warranted to clarify the contribution of individual phytochemicals and validate the molecular pathways responsible for the antioxidant and collagen-promoting activities observed in this study.

CONCLUSION

This study identifies the ability of C. lacryma-jobi extracts to protect against oxidative stress in cells. We demonstrate that cooked adlay extracts containing high levels of coixol (6.51 ± 0.22 µg/g extract), flavonoids, phenolics, and triterpenoids can reduce intracellular ROS. Furthermore, the combination of 200 µM L-ascorbic acid and 50 µg/mL of cooked adlay extract can promote collagen in fibroblast cells at a level equivalent to that of 1,000 µM L-ascorbic acid. This indicates that cooked adlay extracts, even when used alone or in combination with Vitamin C, warrant further investigation before use as a food supplement regimen or as an active ingredient in cosmetic formulations to promote collagen synthesis. Moreover, through our calculations, we determined that 100 grams of raw and cooked adlay were equivalent to 2.50 and 2.49 grams of raw and cooked adlay crude extracts, respectively. These amounts are similar to what is typically used in the recipe.

ACKNOWLEDGEMENTS

The authors would like to acknowledge Prof. Zhi-Hong Jiang and Mr. Khemmachat Pansooksan for their kind help on UHPLC-MS experiment.

AUTHORS’ CONTRIBUTIONS

Woranaree Theangyoo: Writing – Original Draft (Lead), Methodology (Supporting), Validation (Supporting), Investigation (Supporting), Data Curation (Supporting), Writing – Review & Editing (Supporting); Supawadee Parhira: Conceptualization (Supporting), Methodology (Equal), Validation (Lead), Investigation (Equal), Data Curation (Lead), Writing – Review & Editing (Equal), Resources (Lead); Metawee Srikummool: Conceptualization (Lead), Methodology (Lead), Validation (Equal), Investigation (Lead), Data Curation (Equal), Writing – Review & Editing (Lead), Resources (Equal), Supervision (Lead).

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ETHICAL APPROVAL

This research was approved by Naresuan University Institute Review Board No. P10183/63.

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Supplementary materials

Phytochemicals contents determination

Determination of total flavonoids content

Total flavonoid contents of CLR and CLC were determined by using a protocol suggested previously with slight modification (Baba and Malik, 2015). Briefly, the sample solution (50 mg/mL in dimethyl sulfoxide, 125 µL) was mixed with water (2,250 µL) and 5.0 %w/v Sodium nitrile solution (150 µL) then vigorously mixed and incubated for 5 min at room temperature (27 ± 3 °C). The 10% w/v of Aluminum chloride solution (150 µL) was added into the reaction mixture then allowed to stand for 10 min at the room temperature (27 ± 3 °C) before adding 1M Sodium hydroxide (1,000 µL) and water (1,325 µL). The reaction mixture was incubated for 15 min and measuring the absorbance at 510 nm. The total flavonoids content was calculated from a calibration curve of rutin (Sigma-Aldrich, USA) at the concentrations in range of 0 – 75 µg/ml (Y=0.0101X-0.0244, where Y and X represented the absorbance of rutin at 510 nm and the concentrations of rutin in the unit of µg/mL, respectively, and R² = 0.9900), and the result was expressed as milligram rutin equivalent per gram extract (mg RTE/ g extract).

Determination of total phenolics content

The method used in this study was slightly modified from previous report (Pekal and Pyrzynska, 2014). The sample solution (50 mg/ml in dimethyl sulfoxide, 100 µL) was mixed with 10% Folin-Ciocalteu reagent (LOBA Chemie, India, 250 µL) and water (1,150 µL). The mixture was thoroughly mixed for 2 min then added 20%w/v Sodium carbonate solution (1,500 µL) and incubated for 2 h in the dark place at the ambient temperature (27 ± 3 °C). The reaction mixture was then added with water (2 mL) and thoroughly mixed for 2 min. The solution was measured by UV/Vis spectrophotometer (Shimadzu, Japan) at the wavelength of 765 nm. Gallic acid (Sigma-Aldrich, USA) at the concentrations between 0 – 18 µg/ml (Y=0.0942X+0.0929, where Y and X represented the absorbance of gallic acid at 765 nm and the concentrations of gallic acid in the unit of µg/mL, respectively, and R² = 0.9925) was used to perform the standard curve. The total phenolics contents of CLR and CLC were expressed in milligram gallic acid equivalent per gram extract (mg GAE/ g extract).

Determination of Total triterpenoids content

The determination of total triterpenoid contents of CLR and CLC samples was performed according to the aforementioned method suggested by Wei et al. (2015) with some modifications. In brief, the sample solution (1.25 mg/ ml in dimethyl sulfoxide, 600 µl) was thoroughly mixed with 5% vanillin-acetic acid solution (1.0 ml) and sulfuric acid (2.0 ml). The reaction mixture was incubated at 70°C for 30 min and then cooled down to the ambient temperature (27 ± 3 °C). Glacial acetic acid (1.0 ml) was added. The absorbance of sample solutions was measured at 573 nm. The standard curve was done by using ursolic acid standard (Tokyo Chemical, Japan, concentrations of 0–50 µg/ml, Y=0.0414X-0.0541, where Y and X represented the absorbance of usolic acid at 573 nm and the concentrations of ursolic acid in the unit of µg/mL, respectively, R² = 0.9947). Total triterpenoids contents of the extracts were expressed in milligram ursolic acid equivalent per gram extract (mg UAE/ g extract).

Raw data for Figure 3 (Percentage of fibroblast cell viability after being treated with Coix lacryma-jobi L. extracts at various concentrations)

Table S1A. CLR treatment (0-800 µg/ml).

Table S1B. Percentage of fibroblast cell viability after being treated with CLR (0-800 µg/ml).

Table S2A. CLC treatment (0-800 µg/ml).

Table S2B. Percentage of fibroblast cell viability after being treated with CLC (0-800 µg/ml).

Table S3A. CLR treatment (400-1,600 µg/ml).

Table S3B. Percentage of fibroblast cell viability after being treated with CLR (400-1,600 µg/ml).

Table S4A. CLC treatment (400-1,600 µg/ml).

Table S4B. Percentage of fibroblast cell viability after being treated with CLC (400-1,600 µg/ml).

Raw data for Figure 4 (Figure 4. Effects of adlay extracts in preventing H₂O₂-induced cytotoxicity in the fibroblast cells) Cell viability was measured by MTT assay.

Table S5A. Pretreatment the extracts (n=1).

Table S5B. Pretreatment the extracts (n=2).

Table S5C. Pretreatment the extracts (n=3).

Table S5D. Percentage of fibroblast cell viability after being pretreated with the extracts.

Raw data for Figure 5. (Percentage of DCF positive after pre-treatment with the adlay extracts and exposure to H₂O₂-induced oxidative stress in the fibroblast cells.)  ROS level was measured by DCFH-DA assay.

Table S6A. the extract treatment.

Table S6B. Percentage of fibroblast Fluorescent intensity after being treated with the extracts.

Raw data for Figure 7. (Collagen content in fibroblast cells after treatment with the adlay extracts for 72 hr) Collagen content was measured by the Sirius Red/Fast Green Collagen Staining Kit.

Table S7. The extract treatment.

Table S8A. Raw data of MTT assay of the HFF-1 cell viability after H₂O₂ treatments at A₅₇₀.

Table S8B. Percentage of fibroblast cell viability after H₂O₂ treatments.