High-resolution mass spectra (HRESIMS) were measured with an LC/MS-IT-TOF spectrometer. Optical rotations were detected by a JASCO P-1020 digital polarimeter. 1D and 2D NMR spectra were measured with an Advance III-600 instrument. Thin-layer chromatography (TLC) analysis was performed on silica gel plates by spraying with 10% H₂SO₄. A Shimadzu LC-CBM-20 system with an Agilent XDB-C₁₈ column was used for high-performance liquid chromatography (HPLC) purification.

The fruits of Amomum tsao-ko Crevost et Lemarie were collected from Nujiang, Yunnan Province of China in 2022, and were authenticated by Yi Yang (Nujiang Green Spice Industry Research Institute). A voucher specimen (No. 20220701At) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

The dried and ground fruits of A. tsao-ko (1 kg) were extracted with petroleum ether (PE) at room temperature twice (12 L each). The combined extraction was evaporated under vacuum, and separated through silica gel CC, employing ethyl acetate (EtOAc)-PE system (from 2:98 to 50:50, v/v). The EtOAc-PE (5:95, v/v) fraction was separated by Sephadex LH-20 CC [methanol (CH₃OH)-chloroform (CHCl₃), 50:50, v/v] and HPLC [C₁₈ column, acetonitrile (CH₃CN)-H₂O (75:25, v/v)] to yield 1 (4 mg), 2 (3 mg) and 3 (0.5 mg).

(2E,4E,8Z,11Z)-6R-Hydroxytetradeca-2,4,8,11-tetraenal (1):

Pale yellow oil. [α]_D²⁴ −5.45 (c 0.11, MeOH); ¹H and ¹³C NMR (DEPT), see data on Table 1; HRESIMS m/z 221.1521 Da ([M + H]⁺, calculated for C₁₄H₂₁O₂, 221.1536 Da, −1.5 mDa); UV (MeOH) λ_max (log ε): 272 nm.

(2E,4E,8Z)-6R-Hydroxytetradeca-2,4,8-trienal (2):

Pale yellow oil. [α]_D²¹ −3.78 (c 0.09, MeOH); ¹H and ¹³C NMR (DEPT), see data on Table 1; HRESIMS m/z 223.1700 Da ([M+H]⁺, calculated for C₁₄H₂₃O₂, 223.1693 Da, +0.7 mDa); UV (MeOH) λ_max (log ε): 273 nm.

(2E,4E)-6R-Hydroxytetradeca-2,4-dienal (3):

Pale yellow oil. [α]_D²⁴ −6.60 (c 0.02, MeOH); ¹H and ¹³C NMR (DEPT) see data on Table 1; HRESIMS m/z 225.1835 Da ([M+H]⁺, calculated for C₁₄H₂₅O₂, 225.1849 Da, −1.4 mDa); UV (MeOH) λ_max (log ε): 272 nm.

Enzyme inhibition assays followed the previous method with minor modifications [27,28]. In the PTP1B inhibition assay, 100 mL of working buffer containing 3-(N-morpholino)propanesulfonic acid (MOPS, 722.0 mg), dithiothreitol (DTT, 30.0 mg), ethylene diamine tetraacetic acid (EDTA, 25.7 mg) and NaCl (12.1 g) was prepared before the test. Tested samples (10 μL), working buffer (70 μL), and PTP1B enzyme (10 μL) were pipetted into 96-well plates, followed by an incubation for 15 min. After adding the substrate (10 μL), the mixture was incubated for 30 min. To the mixture, 100 μL of Na₂CO₃ was added, and the absorbance was recorded on a FlexA-200 microplate reader. In the GPa inhibitory assay, tested samples (10 μL) and enzyme (50 μL) were added into 96-well plates, and incubated at 37°C for 15 min. After the addition of substrate solution (40 μL), the mixture was incubated for 30 min. Then, the mixture was supplemented with 150 μL of HCl containing ammonium molybdate (10 mg/mL) and malachite green (0.38 mg/mL). After incubation for 20 min, the absorbance was recorded at 620 nm. Sodium orthovanadate (Na₃VO₄) and CP-91149 were applied as the respective positive controls.

Compound 1 had a molecular formula of C₁₄H₂₀O₂ which was assigned by HRESIMS at m/z 221.1521 Da ([M + H]⁺, calculated for C₁₄H₂₁O₂, −1.5 mDa), with five degrees of unsaturation. Its ¹³C NMR (DEPT) data exhibited 14 carbons ascribed to an aldehyde group, eight sp² methines, one oxygenated methine, three methylenes, and a methyl. In the ¹H NMR spectrum, an aldehyde proton at δ_H 9.57, two trans-double bonds at δ_H 6.17 and 7.11 (J = 15.3 Hz) and δ_H 6.56 and 6.28 (J = 15.3 Hz), two cis-double bonds at δ_H 5.41 and 5.62 (J = 10.5 Hz) and δ_H 5.29 and 5.41 (J = 10.5 Hz), and a methyl in triplet at δ_H 0.97, were recognized.

The ¹H and ¹³C NMR data of 1 showed high similarity with lycocasuarinen acid E [29], except that a carboxyl group and two trans-double bonds in lycocasuarinen acid E were changed to be an aldehyde group and two cis-double bonds in 1. With the aid of the ¹H-¹H COSY experiment, the consecutive correlations from H-1 to H-14 established the C₁₄ chain. Moreover, the ROESY correlations of H-1 with H-3, H-3 with H-5, and H-2 with H-4 verified the E-form of Δ^2,4-diene; the correlations of H-7 with H-10, and H-10 with H-13 verified the Z-form of Δ^8,11-diene (Fig. 2).

The stereochemistry of C-6 was determined to be R by its negative rotation ([α]_D²⁴ −5.45), opposite to (2E,4E)-6S-hydroxyldodeca-2,4-dienoic acid ([α]_D²⁰ +11.7) [29]. Hence, compound 1 was characterized as (2E, 4E, 8Z, 11Z)-6R-hydroxytetradeca-2,4,8,11-tetraenal (1).

Compound 2 was assigned the molecular formula of C₁₄H₂₂O₂ according to its HRESIMS at m/z 223.1700 Da ([M+H]⁺, calculated for C₁₄H₂₃O₂, +0.7 mDa), showing four degrees of unsaturation. The NMR data of 2 are similar to 1 besides the absence of one cis-double bond and two extra methylenes. In the ¹H-¹H COSY spectrum, the consecutive correlations from H-1 to H-14 verified the Δ^2,4,8-triene pattern in the structure. Thus, compound 2 was suggested to be the 11,12-dihydro derivative of 1. The Δ²- and Δ⁴-double bonds were deduced as trans-form by the coupling constant of 15.3 Hz, and the Δ⁸-double bond was deduced as cis-form by the coupling constant of 10.5 Hz. This deduction was further confirmed by the ROESY correlations of H-1/H-3/H-5, H-2/H-4, and H-7/H-10. Similarly, the configuration of C-6 was assigned to be R by the negative rotation (−3.78). Thus, compound 2 was identified as (2E,4E,8Z)-6R-hydroxytetradeca-2,4,8-trienal (2).

Compound 3 had a molecular formula of C₁₄H₂₄O₂ which was deduced from the HRESIMS at m/z 225.1835 Da ([M+H]⁺, calculated for C₁₄H₂₅O₂, −1.4 mDa), with two more hydrogens (H) than 2. Compared with 2, the ¹H and ¹³C NMR data suggested the presence of two extra methylenes but the absence of one cis-double bond. From the above features, compound 3 was proposed to be the 8,9-dihydro derivative of 2, and was further supported by the ¹H-¹H COSY correlations from H-1 to H-14. The configuration of Δ^2,4-diene was settled as E by the coupling constant of 15.2 Hz. Its absolute configuration was determined to be 6R by the negative rotation (−6.60). Finally, compound 3 was deduced as (2E, 4E)-6R-hydroxytetradeca-2,4-dienal (3).

PTP1B and GPa are two potential therapeutic targets for the treatment of diabetes. PTP1B as a hydrolase located on the cytoplasmic surface of the endoplasmic reticulum (ER) is widely expressed in insulin-target tissues [30]. By inducing the dephosphorylation of phosphorylated insulin receptor and insulin receptor substrate 1 (IRS1), PTP1B terminates IR signaling and inhibits glucose uptake, thus resulting in insulin resistance [31]. Also, PTP1B can dephosphorylate and inactivate the leptin receptor-associated kinase JAK-2, thereby blocking the signal transduction of leptin [32]. Glycogen phosphorylase (GP) is the key enzyme in catalyzing the rate-limiting step of glycogen degradation. GPa is the active form of GP, inhibiting which can significantly lower the fasting plasma glucose in type 2 DM patients [33,34]. To assess their hypoglycemic potency, compounds 1 and 2 were tested for the PTP1B and GPa inhibitory activity. In Table 2, compounds 1 and 2 displayed moderate inhibition against PTP1B with inhibition rates of 40.2% ± 1.0%, 50.3% ± 0.6% at 200 µM, and 33.8% ± 1.6%, 41.1% ± 2.6% at 100 µM. For the GPa inhibitory assay, compound 1 exhibited high activity with inhibition rates of 94.8% ± 0.2% (200 µM) and 89.8% ± 2.9% (100 µM), whereas compound 2 was inactive at the tested concentrations. The IC₅₀ value of 1 on GPa was calculated as 31.7 μM by a dose-response study (Fig. 3). Compound 3 was not tested for inhibitory activity due to the inadequate amount.

The fruits of A. tsao-ko (Cao-Guo) are widely used as both medicinal materials and food additives in China, showing diverse pharmacological activities such as antibiotic, anti-inflammatory, anticancer, antidiabetic, and neuroprotective effects [11]. Previously, the extracts of A. tsao-ko were reported to have α-amylase and α-glucosidase inhibitory activity in vitro [35] and hypoglycemic activity in vivo [12].

To search for antidiabetic clues from Zingiberaceae plants, our previous study reported a series of flavanol-fatty alcohol hybrids, flavanol-monoterpenoid hybrids, and diarylheptanoids which showed PTP1B and α‑glucosidase inhibitory activity from the EtOAc part of A. tsao-ko [20–24]. Although the low-polarity volatile oils in A. tsao-ko had been revealed with antibiotic, cytotoxic, and antioxidant effects, their antidiabetic potency was still unclear [11]. In connection with our previous research, the petroleum ether soluble part was further studied, from which three new hydroxytetradecenals were obtained and fully determined by HRMS, NMR, and [α]_D data. Their effects against two diabetes-related enzymes PTP1B and GPa were assayed in vitro. Two compounds (1 and 2) exhibited moderate inhibition on PTP1B, and compound 1 showed obvious inhibition on GPa. This study further enriched the antidiabetic constituents of A. tsao-ko.