Abstract Job’s tears (Coix lacryma-jobi L., Poaceae) seeds have been traditionally used for their hypoglycemic effects, but the phytochemicals and underlying mechanisms remain unclear. This study investigates the phytochemicals and inhibitory effects against α-amylase and α-glucosidase of the raw and cooked seeds. The seeds were extracted with 95% ethanol, then fractionated with dichloromethane, ethyl acetate, and water to obtain fractions designated RE, RD, RA, RW (raw seeds), and CE, CD, CA, and CW (cooked seeds), respectively. The phytochemical content, coixol content, and effects on α-amylase and α-glucosidase activities were determined using colorimetry, high-performance liquid chromatography, and enzymatic assays, respectively. The extracts yielded phenolic compounds (4.46-18.84 mg gallic acid equivalents/g), flavonoids (6.21-69.60 mg rutin equivalents/g), and coixol (0.0-30.70 µg coixol/g), with the highest levels of those compounds found in CA, RW, and CE, respectively. Since the tested extracts showed stronger inhibitory effects on α-glucosidase (48.94-98.90%, at 0.1 mg/mL) than on α-amylase (0.30-45.67%, at 5.0 mg/mL), the half-inhibitory concentration (IC₅₀) values were determined only for the potent fractions (RE, RD, RA, CD, and CA) against α-glucosidase. The IC₅₀ values of selected fractions (17.92 to 86.34 µg/mL) were significantly lower than that of acarbose (461.10 µg/mL). Among these, the RA fraction showed the highest efficacy and potency. Flavonoid content correlated strongly with α-glucosidase inhibition, while phenolics and coixol correlated moderately. Dichloromethane and ethyl acetate fractions from raw or cooked Job’s tears seeds, rich in phytochemicals, displayed potential for further investigation of their antihyperglycemic activity in cell-based experiments or in an animal model.

Keywords: Coix lacryma-jobi L., α-amylase, Diabetes mellitus, phenolic, flavonoid

Funding: This research was financially supported by the National Science Research and Innovation Fund (NSRF) of Thailand [Grant NO. R2567B033], Thailand, partially supported by the Global and Frontier Research University Fund, [Grant number. R2567C003] and the Frontier Research and Innovation Cluster Fund, [Grant number R2569C006], Naresuan University, Thailand. The authors are grateful to the Faculty of Pharmaceutical Sciences for funding the undergraduate thesis. The funders played no part in study design, data collection and analysis, publication decisions, or manuscript writing.

Citation: Lohitnavy, O., Pansooksan, K., Phitaknitiwoot, N., Onjek, U., Phoowanatdechawut, T., Kaewchompoo, S., Pekthong, D., Srisawang, P., and Parhira, S. 2026. Total phenolic, flavonoid, and coixol contents, and in vitro α-amylase and α-glucosidase inhibitory activities of Coix lacryma-jobi L. seed extracts. Natural and Life Sciences Communications. 25(4): e2026089.

Graphical Abstract:

INTRODUCTION

Postprandial glycemic control is one of the key targets in diabetes management. One validated nutritional and pharmacological approach is the inhibition of intestinal α-amylase and α-glucosidase (Cheng et al., 2024) to slow starch hydrolysis and attenuate glucose absorption. Plant-derived inhibitors are of interest because they can offer efficacy with favorable safety profiles (Zhang et al., 2024). Coix or Job’s tears (Coix lacryma-jobi L., an accepted name, WFO (2026)), a cereal in the Poaceae family widely cultivated across Asia, is consumed as food and used in traditional medicine (Weng et al., 2022; Kaur et al., 2025). Dehulled seeds are incorporated into beverages, noodles, and health snacks in China, Japan, Sri Lanka, and Thailand (Lin et al., 2022; Shahrajabian et al., 2022). China and Thailand are major producers, supporting their role as main sources of processed ingredients (Rupitak et al., 2024). Nutritionally, coix seeds contain high levels of carbohydrates and proteins (Soni et al., 2023; Kaur et al., 2025). Phytochemical investigations have identified diverse secondary metabolites, including phenolics, flavonoids, tannins, alkaloids, triterpenoids, phytosterols, and benzoxazinoids such as coixol, many of which exhibited antioxidant and other bioactivities (Zhu, 2017; Diningrat et al., 2021; Yahya et al., 2024). Furthermore, pharmacological studies have reported anti-diabetic (Hafizur et al., 2015; Hameed et al., 2019; Li et al., 2023; Mahmood et al., 2025), anti-inflammatory (Hu et al., 2020; Gharegezloo et al., 2024), anti-hyperlipidemic (Zhu et al., 2025), and anti-cancer properties of coix seeds (Weng et al., 2022; Kaur et al., 2025; Parhira et al., 2025; Zhang et al., 2025).

In vitro enzymatic assays are widely utilized to screen plant extracts for diverse biological properties, including antioxidant, anti-tyrosinase, and anti-acetylcholinesterase activities (Kurt-Celep et al., 2023; JA Musa et al., 2025; Yagi et al., 2025). Moreover, α-amylase and α-glucosidase inhibition assays are standard methods for evaluating antidiabetic potential, as demonstrated in various plants such as Phlomis species (Yagi et al., 2025), Centaurea stapfiana (Zengin et al., 2024), Sauauria vulcani Korth. (JA Musa et al., 2025), and Persicaria odorata (Thongra-ar et al., 2021). Evidence for glycemic control has been reported in vivo, extracts of raw coix seeds and purified polysaccharides (coixan A–C) lowered blood glucose in normoglycemic and chemically induced diabetic rodent models (Yin et al., 2019). Prolamins in coix seeds also exhibited hypoglycemic effects, as determined by molecular docking analysis (Li et al., 2023). Nonetheless, the specific mechanisms underlying postprandial control, particularly inhibition of intestinal α-amylase and α-glucosidase, remain insufficiently characterized in coix seed preparations (Taquiqui et al., 2021). Food processing and extraction conditions can substantially alter phytochemical composition and bioactivity (Li et al., 2024; Yagi et al., 2025). Thermal treatment (cooking) disrupts cell walls, gelatinizes starch, and may transform phenolics; solvent partitioning can enrich distinct chemical classes (e.g., ethyl acetate for mid-polarity phenolics) (Li et al., 2024). Despite the predominance of cooked coix in the diet, a systematic, side-by-side evaluation of raw (uncooked) versus cooked seeds, combined with solvent fractionation, chemical standardization (including coixol quantification), and α-amylase/α-glucosidase inhibition and antioxidant activity, has been limited.

Therefore, this study prepared crude ethanolic extracts of raw and cooked C. lacryma-jobi L. seeds and partitioned them with dichloromethane, ethyl acetate, and water. We quantified total phenolics (gallic acid equivalents, GAE) and flavonoids (rutin equivalents, RTE) and determined coixol content using high-performance liquid chromatography (HPLC). The in vitro α-amylase and α-glucosidase enzymatic assays were performed to evaluate the antihyperglycemic activity of the coix seed extract. The results establish a chemically standardized basis for selecting coix seed extracts with potential application in dietary strategies for managing postprandial glycemia.

MATERIALS AND METHODS

Materials

Standard gallic acid was purchased from Sigma-Aldrich, China. Standard reagents, including dimethyl sulfoxide (DMSO), α-amylase from porcine pancreas, α-glucosidase from Saccharomyces, sodium phosphate monobasic, starch, and 3,5-dinitrosalicylic acid (DNS), were purchased from Sigma Aldrich, USA. Acarbose hydrate was bought from Tokyo Chemical Industry Co., Ltd, Japan. Potassium sodium (+) tartrate was purchased from Elago Enterprises Pty Ltd, Australia. p-Nitrophenyl-α-D-glucopyranoside (pNPG) was bought from EMD Millipore Corp, USA. Folin-Ciocalteu’s phenol reagent was bought from Merck, Germany. Aluminum chloride hydrated, di-sodium hydrogen orthophosphate anhydrous, and sodium hydroxide were purchased from Ajax Finechem, Australia. Deionized water, absolute ethanol, dichloromethane, and ethyl acetate were purchased from LabScan Co., Ltd., Thailand. 95% Ethanol was obtained from the Liquor Distillery organization, Thailand. Coixol reference standard (purity 98.78%) was purchased from MedChemExpress, NJ, USA. The 96-well plates were bought from SPL, Korea.

General instruments

Eppendorf Thermomixer comfort, heating microplate shaker (Topscien, China), magnetic stirrer (Conversant technology, Thailand), microplate reader BioTek, Synergy H1 hybrid reader (BioTek, USA), pH meter (pH 700, Eutech instruments, Singapore), Vortex-genie 2 (Scientific industries, USA), ultrasonic cleaning equipment (Shenzhen Jietai Ultrasonic Cleaning Equipment Co., Ltd., China), and rotary evaporator (Buchi, Switzerland) were used to perform relevant experiments.

Sample preparation

Dried raw seeds (Figure 1A) of Job’s tears (C. lacryma-jobi L.) were obtained from Thanya Farm Co., Ltd. (Nonthaburi, Thailand). They were authenticated and described in our previous report (Parhira et al., 2025), with voucher reference no. 005348. The cooked seeds (Figure 1B) were prepared using 500 g of raw seeds and 1.5 L of deionized water in an automatic rice cooker (Phillips, Bangkok, Thailand). The cooked seeds were further dried in a hot air oven (50°C), then blended, and the process was repeated 6 times to obtain a total of 3 kg of cooked seeds. The raw seed powder was prepared directly using a blender. The raw or cooked seed powders (3 kg) were macerated with 15 L of 95% ethanol and sonicated for 30 min. The mixture was filtered to separate the plant residue and ethanolic filtrate. The plant residue was re-extracted twice under the same conditions. The filtrates from three extraction cycles were concentrated under reduced pressure using a rotary evaporator to yield crude ethanolic extracts of raw (RE) and cooked (CE) seeds. The crude extracts (RE or CE; 60 g) were then subjected to liquid–liquid partitioning by dispersing in 200 mL of water, then fractionated with dichloromethane (400 mL, three times) in a separatory funnel. The dichloromethane layer was collected, and the remaining aqueous layer was subsequently partitioned with ethyl acetate (400 mL, three times). Organic layers were concentrated at 45°C under reduced pressure; the final aqueous layer was freeze-dried. Fractions obtained from the dichloromethane, ethyl acetate, and water layers of the raw seeds were abbreviated as RD, RA, and RW, while those from the cooked seeds were labeled CD, CA, and CW, respectively. Percent yields (% w/w) of the samples were calculated by comparing the sample weights to 100 g of dry plant. All dried extracts/fractions were kept in sealed amber vials, protected from light, and stored at –20°C. Working solutions were freshly prepared in appropriate solvents before assays.

Figure 1. Appearance of C. lacryma-jobi L. raw seeds (A), and cooked seeds (B).

Phytochemical measurement

Total phenolic content

Total phenolic content was determined following the method previously reported (Srisawang et al., 2024). Briefly, 25 µL of extract (2 mg/mL in methanol) was mixed with 100 µL of Folin Ciocalteu’s reagent (diluted 1:4 v/v with distilled water) in a flat-bottom 96-well plate. The mixture was shaken (200 rpm, 1 min), then 75 µL of saturated sodium hydrogen carbonate (6% w/v) was added and shaken for another 1 min. The plates were incubated at 25 ± 2 °C in the dark for 2 hours. Absorbance was read at 765 nm using a microplate reader. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/ g extract (mean ± standard deviation (SD), n=3).

Total flavonoid content

Total flavonoid content was determined according to a previously reported method (Srisawang et al., 2024) with some modifications. Briefly, in a 96-well plate, 50 µL of extract (1 mg/mL in methanol) was mixed with 10 µL of a 10% (w/v) aluminum chloride solution in methanol, 150 µL of methanol, and 10 µL of 1 M sodium acetate. After incubation at 25 ± 2 °C in the dark for 40 min, absorbance was measured at 415 nm. Total flavonoid content was expressed as mg rutin equivalents (RTE)/ g extract (mean ± SD, n=3).

High-performance liquid chromatographic (HPLC) quantification of coixol

The coixol content in eight C. lacryma-jobi L. seed extracts (RE, RD, RA, RW; CE, CD, CA, CW) was quantified following a reported protocol (Parhira et al., 2025) with slight modifications. Sample solutions were prepared at 5.0 mg/mL in methanol; the coixol reference standards were prepared in methanol for calibration. Before injection, solutions were clarified by centrifugation and filtered through 0.45 µm filters. Analyses were performed on a Shimadzu LC-10ATvp HPLC system equipped with a C₁₈ column (ACE^® Excel 5 C₁₈, 250 mm × 4.6 mm, 5 µm). The mobile phase was acetonitrile:  0.1% (v/v) phosphoric acid in water (35:65, isocratic) at a flow rate of 0.8 mL/min. The column temperature was 25°C. The injection volume was 20 µL, and detection was performed at 230 nm. Total run time was 20 min. Calibration curves for coixol (0.05-1.0 µg/mL) were used to calculate coixol contents in the extracts. Results were reported as µg coixol per g of extract and expressed as mean ± SD from three independent experiments.

In vitro antihyperglycemic assays

In vitro α-amylase inhibition assay

The method was adapted from the previous protocol (Telagari and Hullatti, 2015) with minor modifications. In a microcentrifuge tube, a reaction mixture containing the 50 μL of the tested samples (to reach the final concentration of 5 mg/mL) and 100 μl of α–amylase (0.625 mg/mL) was preincubated at 37°C for 20 min, then 100 μL of 1% soluble starch as a substrate was added and continually incubated at 37°C for 30 min. The 100 μL of 3,5-dinitrosalicylic acid (DNS) color reagent was then added, and the mixture was boiled at 90°C for 10 min to stop the reaction. The reaction mixture was allowed to cool down to room temperature. The absorbance of the resulting mixture was measured at 540 nm using a microplate reader. Acarbose at a concentration of 0.1 mg/ml was used as a positive control. In the absence of test samples, a vehicle control was established in parallel, and each experiment was performed in triplicate. The results were expressed as mean ± SD of the percentage inhibition, which was calculated using the formula,

Inhibitory activity (%) = (1 – Absorbance sample_540nm/Absorbance control_540nm) ×100

In vitro α-glucosidase inhibition assay

The method was modified from the previous report (Promyos et al., 2017). Briefly, 50 µL of sample solution (in DMSO with various concentrations of 10-100 µg/mL) was mixed with 50 µL of α-glucosidase (0.01 U/mL) in a 96-well plate. The reaction mixture was incubated at 37°C for 15 min, then p-nitrophenyl-α-D-glucopyranoside (pNPG, 1.25 mM) was added, and the mixture was incubated at 37°C for an additional 40 min after which the absorbance at 405 nm was measured using a microplate reader. Acarbose (10.0 mg/mL) and DMSO were used as the positive and vehicle controls, respectively. The results were expressed as mean ± SD of the percentage inhibition, which was calculated using the formula,

Inhibitory activity (%) = (1 – Absorbance sample_405nm/Absorbance control_405nm) ×100

The concentration–response curves were fitted using nonlinear regression to estimate the IC₅₀. Each assay was performed in triplicate.

Statistical analysis

The mean ± SD values for the tested samples from three independent experiments are presented. Data were analyzed by one-way analysis of variance (ANOVA), and then the Bonferroni post hoc adjustment was performed manually by calculating pairwise comparisons and adjusting the significance level (α (new) = α / k, where k is the number of comparisons).  All analyses were performed using the Data Analysis Toolpak in Microsoft Excel, with statistical significance set at P < 0.05. Pearson’s correlation analysis (n = 5) was performed using Microsoft Excel to explore the relationship between bioactive compounds (phenolic, flavonoid, and coixol) and α-glucosidase inhibitory activity (IC₅₀). The analysis is descriptive and exploratory, aimed at observing general trends. The absolute magnitude of the observed correlation coefficient was then interpreted as recommended by Schober et al. (2018).

RESULTS

Physical appearance and percentage yield of the extracts from C. lacryma-jobi L. seeds

The crude ethanolic extracts, dichloromethane fraction, and ethyl acetate fraction of C. lacryma-jobi L. seeds, both raw and cooked, exhibited similar physical characteristics, appearing as a mixture of yellowish wax and liquid, whereas the water fractions were yellowish powders. The extraction yield of the ethanolic crude extract from raw seeds (RE, 5.89%) was approximately twice that of cooked seeds  (CE, 2.92%). However, the raw and cooked seed dichloromethane (RD, 0.40%; CD, 0.75%), ethyl acetate (RA, 0.09%; CA, 0.05%), and water (RW, 1.06%; CW, 0.33%) fractions resulted in very low yields, as shown in Table 1.

Table 1. Physical appearance and percentage yields (%) of the extracts from raw and cooked C. lacryma-jobi L. seeds.

Note: RE: Ethanolic crude extract of raw coix seed; RD: Dichloromethane fraction of raw coix seed; RA: Ethyl acetate fraction of raw coix seed; RW: Water fraction of raw coix seed; CE: Ethanolic crude extract of cooked coix seed; CD: Dichloromethane fraction of cooked coix seed; CA: Ethyl acetate fraction of cooked coix seed; CW: Water fraction of cooked coix seed.

Phytochemical compositions 

The phytochemical contents of C. lacryma-jobi L. seed extracts varied across organic solvents used for fractionation and between raw and cooked seeds, as shown in Figure 2A–C. For total phenolic content (Figure 2A), the cooked ethyl acetate fraction (CA) showed the statistically highest level (18.84 mg GAE/g extract) among all tested samples. For total flavonoid content (Figure 2B), the raw aqueous fraction (RW) exhibited the statistically highest level (69.60 mg RTE/g extract) among all fractions from raw seeds, while the cooked ethyl acetate fraction (CA, 55.85 mg RTE/g extract) exhibited the highest amount among the fractions from the cooked seeds. In contrast, the raw ethanol extract contained the lowest flavonoid content (6.21 mg RTE/g extract). For coixol content (Figure 2C), the cooked ethanol extract (CE) had the highest amount (30.70 µg/g extract) among all tested fractions, followed by the dichloromethane fraction from cooked seeds (26.26 µg/g extract). The dichloromethane fractions from raw seeds exhibited statistically higher coixol amount (22.74 µg/g extract) than those from raw seeds. Coixol was not detected in the water fractions (RW and CW).

Figure 2. Total phenolic content (A), total flavonoid content (B), and Coixol content (C) of raw and cooked C. lacryma-jobi L. seed extracts.

Note: RE: Ethanolic crude extract of raw coix seed; RD: Dichloromethane fraction of raw coix seed; RA: Ethyl acetate fraction of raw coix seed; RW: Water fraction of raw coix seed; CE: Ethanolic crude extract of cooked coix seed; CD: Dichloromethane fraction of cooked coix seed; CA: Ethyl acetate fraction of cooked coix seed; CW: Water fraction of cooked coix seed.  The symbol “*” indicates the highest values that differ significantly from other values within the same group of raw or cooked seeds at P < 0.05, while the symbol “**” indicates significant differences across all samples at P < 0.05.

In vitro α-amylase inhibitory activity

The α-amylase inhibition activity of the extracts from seeds of C. lacryma-jobi L. is shown in Table 2. It was found that all fractions exhibited weaker activity (0.30-45.67% inhibition at 5.0 mg/mL) than acarbose (66.12% at 0.1 mg/mL). The dichloromethane fractions (RD: 45.67%, CD: 36.68%) exhibited the highest inhibitory activity, followed by the ethyl acetate fractions (RA: 28.20%, CA: 25.57%). Ethanolic extracts (RE: 24.15%, CE: 17.95%) showed only moderate activity, while the water fractions (RW, CW) were almost inactive (<2%). This indicates that non-polar fractions had greater α-amylase inhibitory potential than polar fractions.

Table 2. Inhibitory effects of C. lacryma-jobi L. seed extracts on α-amylase and α-glucosidase enzymes.

Note: RE: Ethanolic crude extract of raw coix seed; RD: Dichloromethane fraction of raw coix seed; RA: Ethyl acetate fraction of raw coix seed; RW: Water fraction of raw coix seed; CE: Ethanolic crude extract of cooked coix seed; CD: Dichloromethane fraction of cooked coix seed; CA: Ethyl acetate fraction of cooked coix seed; RW: Water fraction of cooked coix seed.  The symbol “*” indicates statistically significant differences from Acarbose at P < 0.05.

In vitro α-glucosidase inhibitory activity

The α-glucosidase inhibitory activity is summarized in Table 2. The results indicated that the ethyl acetate (RA, CA) and dichloromethane (RD, CD) fractions from both raw and cooked seeds exhibited the strongest activity, with inhibition exceeding 95% at 0.1 mg/mL. Their IC₅₀ values were remarkably low, ranging from 17.92 µg/mL (RA) to 33.57 µg/mL (RD), indicating potent inhibitory effects. In contrast, ethanolic extracts (RE, CE) showed moderate or undetectable activity, and water fractions (RW, CW) were inactive. Notably, all active fractions exhibited statistically greater α-glucosidase inhibitory activity than the positive control, acarbose (IC₅₀ = 461.10 µg/mL), highlighting their potential for antihyperglycemic activity.

The correlation analysis revealed negative associations between phytochemical contents and IC₅₀ values for α-glucosidase inhibitory activity. Among the tested parameters, flavonoid content showed the strongest negative correlation, followed by coixol content and phenolic content (Table 2). It indicated that higher flavonoid content is associated with a strong increase in α-glucosidase inhibitory activity, whereas coixol and phenolic contents contribute to a moderate degree.

DISCUSSION

The present study revealed that the extraction yields of C. lacryma-jobi L. seeds differed among solvents and between raw and cooked seeds (Table 1). Ethanolic crude extracts from raw seeds yielded a higher percentage than those from cooked seeds. The water, ethyl acetate, and dichloromethane fractions yielded lower percentages (0.05 to 0.75%) but were apparently more concentrated and phytochemical-rich. Cooking by steaming decreased the yields of ethanolic and aqueous fractions compared to raw seeds, indicating thermal degradation or binding of phytochemicals during processing, in agreement with a previous study on thermally treated cereals and legumes (Antony and Farid, 2022).

Phytochemical profiling showed that the ethyl acetate fraction of cooked seeds (CA) contained significantly higher (P < 0.05) levels of phenolic compounds than all other fractions, while RW and CA showed the highest flavonoid contents among all fractions from raw and cooked seeds, respectively. Coixol concentrations in the RE, RD, RA, CE, CD, and CA fractions in this study ranged from 11.47 to 30.70 µg/mL, which were higher than the values (2.33 to 9.69 µg/mL) reported in our previous study (Parhira et al., 2025). Conversely, coixol was not detected in the water fractions (RW and CW), consistent with our earlier findings (Parhira et al., 2025). These discrepancies may be due to differences in coix seed batches and extraction methods. Pearson’s correlation analysis confirmed that flavonoid content (-0.74) exhibited a strong correlation (Schober et al., 2018)., whereas phenolic content (-0.48) and coixol content (-0.52) showed moderate correlations (Schober et al., 2018) with α-glucosidase inhibitory activity (IC₅₀). These results support the essential role of phenolics and flavonoids in contributing to enzyme inhibition, consistent with prior findings that polyphenols interact with the catalytic sites of carbohydrate-hydrolyzing enzymes (Kim et al., 2000; Kim et al., 2015). Interestingly, the CE fraction, despite having the highest coixol content, did not exhibit α-glucosidase inhibitory activity, indicating that the antidiabetic effect of coixol may not occur via this pathway. Previous studies have demonstrated that coixol exhibits exclusive glucose-dependent insulinotropic activity in βTC6 cells, with minimal cytotoxic effects (Hafizur et al., 2015), and potentiates glucose-stimulated insulin secretion via cAMP-mediated signaling pathways (Hameed et al., 2019).

The enzyme inhibition assays clearly distinguished between α-amylase and α-glucosidase activities. For α-amylase, the dichloromethane fractions RD (45.67%) and CD (36.68%) exhibited the highest inhibition, exceeding those of other extracts from raw and cooked seeds, respectively, but were still significantly (P < 0.05) lower than that of acarbose (66.12%). In contrast, α-glucosidase inhibition was remarkably strong in the ethyl acetate (RA: 98.90%, CA: 99.40%) and dichloromethane (RD: 95.93%, CD: 96.22%) fractions, with IC₅₀ values ranging from 17.92 to 33.57 µg/mL. The IC₅₀ value of RE was 86.34 µg/mL. These IC₅₀ values were significantly lower (P < 0.05) than those of acarbose (461.10 µg/mL), supporting the fractions’ potential inhibitory effect on α-glucosidase. The ethanolic extract of cooked seed (CE) and water extracts (RW and CW) exhibited negligible α-glucosidase inhibitory activity, underscoring the importance of moderately polar phytoconstituents.

A recent study on coix fruit extract by Mahmood et al. (Mahmood et al., 2025) reported the total phenolic (5.3 mg GAE/g extract), total flavonoid (1.2 mg quercetin equivalents/g extract) contents, as well as the IC₅₀ values of the aqueous extract of the peel-off coix fruits against α-amylase (11.02 mg/mL) and α-glucosidase (4.31 mg/mL) activities. These data differed from our results for the ethanolic crude extract of raw seed (RE) regarding total phenolic content (4.46 mg GAE/g extract) and total flavonoid content (6.21 mg RE/g extract). Due to the limited solubility in our aqueous assay system, RE exhibited only 24% inhibition against α-amylase at the maximum concentration tested (5.0 mg/mL). However, the coix fruit extract required a significantly higher (2.2 times) concentration (11.02 mg/mL) to reach 50% inhibition. Furthermore, the IC₅₀ value of RE against α-glucosidase (0.086 mg/mL) was 50 times lower than that of the coix fruit extract (4.31 mg/mL), indicating significantly higher potency. These results highlight the critical influence of extraction solvents on the phytochemical profile and subsequent bioactivities of the extracts.

The selective inhibition of α-glucosidase over α-amylase was particularly significant (P < 0.05), suggesting the potential to reduce postprandial hyperglycemia while minimizing gastrointestinal side effects often linked to potent α-amylase inhibition (Kwon et al., 2007). The fact that all active fractions demonstrated significantly greater α-glucosidase inhibitory potency than acarbose supports the antihyperglycemic activity of C. lacryma-jobi L. seeds.

The findings showed that the moderate-polar fractions of C. lacryma-jobi L. seeds, particularly the ethyl acetate and dichloromethane extracts, contained significantly higher levels of phenolic and flavonoid compounds, which contributed to their potent α-glucosidase inhibition. Despite reductions in extraction yields after cooking, these bioactive fractions retained strong enzyme-inhibitory activity, suggesting that the active components remained stable under thermal processing. These α-glucosidase-inhibitory activities of phenolic and flavonoid compounds, alongside the alternative antidiabetic pathway of coixol, a major active constituent of the seed, support the potential of C. lacryma-jobi L. seeds as a promising natural alternative or adjunct treatment for type 2 diabetes.

However, this study investigated enzyme inhibition exclusively in vitro. Since α-glucosidase is a membrane-bound enzyme localized at the microvilli of enterocytes (Rose et al., 2018), the inhibitory efficacy of a compound is not only determined by its binding affinity to the enzyme’s active site but also by its physicochemical properties (e.g., lipophilicity, partition coefficient, molecular weight, and ionization state). These properties can significantly affect its ability to navigate the hydrophilic mucous layer and interact with a hydrophobic lipid bilayer environment. Therefore, our results should be confirmed by cell-based assays such as those using Caco-2 cells, alongside further investigations into their in vivo efficacy and bioavailability. Future work will include isolating the active constituents, evaluating their synergistic effects, and validating their antihyperglycemic activity in animal models or clinical studies.

CONCLUSION

The novelty of this work lies in its parallel evaluation of processing methods (raw and cooked coix seeds) and extraction depths (crude extracts and sub-fractions), providing the first report on how these factors influence phytochemical partitioning and subsequent antihyperglycemic activity. This study demonstrated that the extracts exhibit a targeted inhibitory preference for α-glucosidase over α-amylase, with the most potent fractions (RE, RD, RA, CD, and CA) showing IC₅₀ values significantly lower than those of the standard drug acarbose. Among these, the raw ethyl acetate (RA) fraction appeared to be the most effective. The flavonoid content of the extracts showed a strong correlation with α-glucosidase inhibitory activity, whereas phenolics and coixol content showed moderate correlations. In summary, these findings validate the traditional use of C. lacryma-jobi L. and highlight its dichloromethane and ethyl acetate fractions as highly promising candidates for further development as natural antihyperglycemic agents in cell-based or animal models.

ACKNOWLEDGEMENTS

The authors are grateful to Assistant Professor Pranee Nangngam for authenticating the herbarium specimen of Coix lacryma-jobi L. seeds. The authors thank Dr. David Edward Reed and Mr. Michael John Reed for their assistance with proofreading.

AUTHOR CONTRIBUTIONS

Ornrat Lohitnavy: Conceptualization (Lead), Methodology (Lead), Writing - Original Draft (Lead), Writing - Review & Editing (Lead), Visualization (Lead), Supervision (Lead); Khemmachat Pansooksan: Methodology (Equal), Software (Equal), Validation (Equal), Formal Analysis (Equal), Investigation (Equal), Writing - Original Draft (Supporting), Writing - Review & Editing (Supporting), Visualization (Supporting); Nalinthip Phitaknitiwoot: Methodology (Equal), Investigation (Equal), Writing – Original (Supporting), Visualization (Supporting); Ubonwan Onjek: Methodology (Equal), Investigation (Equal), Writing – Original (Supporting), Visualization (Supporting);  Thatchaya Phoowanatdechawut: Methodology (Equal), Investigation (Equal), Writing - Original, Visualization (Supporting); Sudthiworarak Kaewchompoo: Methodology (Equal), Investigation (Equal), Writing – Original (Supporting), Visualization (Supporting); Dumrongsak Pekthong: Writing - Review & Editing (Supporting), Visualization (Supporting); Piyarat Srisawang: Writing - Review & Editing (Supporting), Visualization (Supporting), Funding Acquisition (Equal), Project Administration (Equal); Supawadee Parhira: Conceptualization (Lead), Methodology (Lead), Software (Lead), Validation (Lead), Formal analysis (Lead), Investigation (Lead), Resources (Lead), Data Curation (Lead), Writing - Original Draft (Lead), Writing - Review & Editing (Lead), Visualization (Lead), Supervision (Lead), Project Administration (Lead), Funding Acquisition (Lead). All authors have read and approved the final manuscript.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

DECLARATION OF AI USE IN MANUSCRIPT PREPARATION

Generative AI and AI-assisted tools (Gemini and Grammarly) were utilized during the drafting process strictly for grammatical corrections. The authors maintained full control over the scientific ideas, data interpretation, and final content.

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