The reference substances of cucurbitacin B (CuB; lot: Z02M7X10137; purity ≥98%), cucurbitacin D (CuD; lot: DSTDH008101; purity ≥98%), and cucurbitacin E (CuE; lot: Z01M7S10309; purity ≥98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Bifendate (lot: 73536-69-3; purity ≥98%) was purchased from Chengdu Must Bio-Technology (Chengdu, Sichuan Province, China) as an IS. All other chemical reagents, including methanol and formic acid (Dikma Technologies, Beijing, China), were of HPLC quality. P. Melo (RP20240313) was purchased from Shaanxi Pioneer Biotech Co., Ltd. And identified by Professor Zhenyue Wang of Heilongjiang University of Chinese Medicine.

An Agilent 6430 triple quadrupole mass spectrometer with an ESI source interface (Agilent Technologies, Santa Clara, CA, United States) was used in conjunction with an Agilent 1290 UHPLC system (Agilent Technologies). A Waters ACQUITY HSS T3 Column (1.8 μm, 2.1 × 100 mm) (Waters, Manchester, UK) was also used for the separations. Gradient elution was performed using water (A) and methanol (B) as mobile phases. The gradient elution procedure was used for 0.0–1.0 min, 80%–80% B; 1.0–1.5 min, 80%–90% B; 1.5–3.0 min, 90%–90% B; 3.0–3.2 min, 90%–80% B; and 3.2–4 min, 80%–80% B. The analytical temperature was 35 °C, and the flow rate was 0.3 mL/min. An injection of 5 μL volume was administered, after which the needle was washed. Positive ion-mode analysis was performed for the samples. The capillary voltage was set to 4500 V, source temperature to 100 °C, and the desolvation temperature to 350 °C (Wang et al., 2014). High-purity nitrogen was used as the nebulizing gas, and nitrogen was used as the drying gas at a flow rate of 11 L/min. The Agilent Mass Hunter workstation was used to gather the experimental data (Wang Z et al., 2023). Using specific precursor and product ion transitions at m/z 581.2→521.9 for CuB, m/z 539.3→480.2 for CuD, m/z 578.9→520.9 for CuE, and m/z 418.9→387.1 for IS at various fragmentor voltage (FV) and collision energy (CE) conditions listed in Table 1, quantification was accomplished using multiple reaction monitoring (MRM) mode. The production mass spectra of the three analytes and IS are displayed in Figure 2.

CuB (4.953 mg), CuD (4.603 mg), and CuE (4.544 mg) were accurately weighed and converted to 10 mL in methanol to prepare a single mother liquor of 0.4953, 0.4603, and 0.4544 mg/mL, respectively. The IS 5.504 mg was dissolved in methanol in a 20 mL volumetric flask, and a 0.275 mg/mL stock solution was obtained. The calibration solution was prepared by using the mother liquor gradient dilution method.

Following the necessary addition of blank plasma and standard solutions to the samples for the standard calibration curves, the final concentrations obtained were 2.109, 4.212, 10.50, 21.04, 42.10, 210.4, and 420.3 ng/mL for CuB; 10.08, 20.21, 50.52, 101.2, 202.7, 404.2, and 808.2 ng/mL for CuD; 2.103, 4.221, 10.54, 21.13, 42.09, 210.3, and 420.1 ng/mL for CuE. These concentrations were achieved by spiking 100 μL of blank plasma with 10 μL of IS and 100 μL of a mixed standard solution containing the three compounds. Drug-free plasma was used to create QC samples at four different concentration levels, HQC are 300.2, 600.3, and 300.8 ng/mL; MQC are 150.1, 300.1, and 150.4 ng/mL; LQC are 5.102, 25.31, and 4.912 ng/mL; LLOQ are 2.109, 10.08, and 2.103 ng/mL for CuB, CuD, and CuE. All working solutions were stored at 4 °C throughout the experiment.

A specific quantity of P. Melo was homogenized, and 8-fold volumes of methanol were added to the sample. The mixture was then subjected to three sequential ultrasonic extractions (for 40 min each) at room temperature. Following each extraction cycle, the slurry was vigorously shaken, filtered through a 0.22 μm membrane filter, and the filtrate was collected. The pooled filtrates were concentrated via rotary evaporation under reduced pressure to obtain the crude P. Melo extract. Simultaneously, a 20% (w/v) PVP K30 solution was prepared by dissolving the polymer in deionized water. The solution was magnetically stirred (600 rpm) at 60 °C in a water bath until a homogeneous, clear aqueous phase was achieved.

A total of 12 male Sprague Dawley (SD) rats (weight: 220 ± 20 g) were provided by the Animal Experiment Center of Heilongjiang University of Traditional Chinese Medicine (Production license number: SYXK (Hei) 2021-010). The Laboratory Animal Ethics Committee of Heilongjiang University of Traditional Chinese Medicine approved the study (Approval No.: 2024053114).

Animals were randomly assigned to the following two experimental groups (n = 6 per group): CUT and MP-NPs. Treatments were administered once daily for seven consecutive days, with the plasma sampling performed at predetermined time points after administration. Dosage was calculated based on human-equivalent doses converted to rat pharmacokinetic parameters using interspecies scaling factors. The human-rat dose conversion procedure was used to determine the dose of CUT per rat to be 0.03 g. The recommended daily dosage for humans is 6 tablets. The dose of MP-NPs per rat was determined to be 0.06 g per rat using the human-rat dose conversion formula, whereas the dose of P. Melo for human usage was 3 g, with reference to the Pharmacopoeia of the People’s Republic of China 2025.

At 0.083, 0.25, 0.50, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h following oral administration of CUT and MP-NPs, 0.25 mL of plasma samples were sampled by rapidly collecting and centrifuging at 4 °C, followed by freezing at −20 °C for further analysis (Yu et al., 2023).

Blank plasma sample (100 μL) was vortexed for 60 s after adding 100 μL of IS. To guarantee complete extraction, 3 mL of dichloromethane was then added to each sample, followed by vortexing for 120 s. Subsequently, the samples were centrifuged for 10 min at 4 °C and 3,500 rpm. At 40 °C, the top organic phase was carefully moved into a sterile tube, dried off with a mild stream of nitrogen gas, and then reconstituted in 100 μL of the original mobile phase. Following a 120 s vortex, the extracts were filtered through a 0.22 μm Millipore filter before subjecting them to quantitative analyses by injecting 5 μL of them into the UHPLC-MS/MS apparatus.

The established UHPLC-MS/MS method was applied to simultaneously measure the plasma concentrations of the three analytes in rats following dose administration. Pharmacokinetic parameters have attracted attention in exploring drug absorption and distribution. These parameters include T _1/2, C _max, T _max, and AUC (Table 6). The average plasma concentration-time curve is presented in Figure 4. The pharmacokinetics of SD rats following oral administration of CUT and MP-NPs were investigated through UHPLC-MS/MS. The plasma concentrations of the three analytes were calculated. The T _max values of these analytes gradually increased. The MP-NPs group reached C _max more rapidly than the CUT group. AUC _0→t and A U C 0 → ∞ also demonstrated that MP-NPs were superior to CUT preparations, suggesting that MP-NPs had higher AUC in vivo, possibly due to the increased drug bioavailability and absorption by the nanoparticles, thereby enhancing drug accumulation in the body.

Mass spectrometric conditions, including ionization and parameters, were first optimized. A higher response was observed in the ESI positive-ion mode than in the negative-ion mode. Mass spectral parameters of the compounds varied based on the instrument and operating conditions (Abdollahi et al., 2024; Bali et al., 2011; Aluri et al., 2023). The MRM mode was employed for testing the precursor and product ions of the three compounds and IS to ensure reliability. Optimal ion transition pairs for quantitation were identified: m/z 581.2→ 521.9 for CuB, m/z 539.3→ 480.2 for CuD, m/z 578.9→ 520.9 for CuE, and m/z 418.9→ 387.1 for IS in the MRM mode. The effects of different mobile phase combinations (acetonitrile–water and methanol–water) and buffers (ammonium formate, formic acid, ammonium acetate, and acetic acid) on chromatographic behavior were examined. The mobile phase selected was methanol–water, with a column temperature and flow rate of 35 °C and 0.3 mL/min, respectively. The injection volume was 5 μL, and the total run time was 4 min.

An appropriate IS with good stability that remains unchanged during sample processing and analysis must be selected for biological sample analysis. An unstable IS may lead to errors in the quantitative analysis. This study explored the effects of m-nitrophenol and bifendate as potential IS. When bifendate was detected in the positive-ion mode, it had a retention time with a significant separation from the analyte, generating symmetrical and sharp peaks, which indicated its stability and sensitivity. Therefore, the bifendate was selected as the IS in the experiment.

Cucurbitacin I (CuI) is a tetracyclic triterpenoid compound primarily found in Cucurbitaceae plants. CuB, CuD, CuE, and CuI all had cucurbitane-type triterpenoid parent nuclei, but exhibited varying substituents, affecting solubility and bioavailability. CuI is well-known for exerting powerful anti-cancer properties, particularly by regulating the JAK/STAT and PI3K/Akt pathways (Xu et al., 2022; Üremiş et al., 2022; Zhu et al., 2018). CuB, CuD, and CuE also exert equally strong anti-cancer, hepatoprotective, and anti-inflammatory effects compared with CuI. In P. Melo, the content of CuB, CuD, and CuE was significantly higher than that of CuI, and pharmacokinetic data on CuB, CuD, and CuE (especially components in TCM nanosuspensions) in P. Melo remain scarce. Our MP-NP formulations specifically address the bioavailability limitations of these understudied cucurbitacins. Therefore, this study primarily investigated the pharmacokinetics of CuB, CuD, and CuE nanosuspensions. The pharmacokinetic studies revealed that MP-NPs were substantially advantageous over CUT in terms of the drug absorption rate, C _max, AUC, and T _1/2, possibly because of the nanosuspension characteristics of MP-NPs (such as larger surface area, higher solubility, and slower drug release). These characteristics can improve drug bioavailability and prolong its action time in the body. For example, the MP-NPs group reached C _max more quickly after absorption than the CUT group (P < 0.05), potentially because of the nanosuspension characteristics of MP-NPs. MP-NPs may have better bioavailability or higher solubility, and so, the drug is more easily absorbed into the bloodstream. For CuD and CuE, the T _1/2 of MP-NPs was generally longer than that of CUT. For instance, the T _1/2 of CuD was 6.967 h (MP-NPs) versus 5.328 h (CUT), and for CuE, it was 7.053 h (MP-NPs) versus 5.991 h (CUT). This suggests that nanosuspension formulations have a longer retention time in the body, likely due to their physicochemical properties, which allow for slow release. AUC values of the three compounds were significantly higher in the MP-NPs group (P < 0.05). AUC _0→t for CuB was 875.5 ng·h/mL (MP-NPs) versus 617.9 ng·h/mL (CUT). It was 1,259 ng·h/mL (MP-NPs) versus 1,033 ng·h/mL (CUT) for CuD and 751.6 ng·h/mL (MP-NPs) versus 602.9 ng·h/mL (CUT) for CuE (Abdollahi et al., 2024; Bali et al., 2011). This suggests that MP-NPs have a higher AUC, likely because their nanosuspension enhances the drug’s bioavailability and absorption rate in the body, thereby increasing drug accumulation.

Because of the polymeric nature of PVP K30, a high-molecular-weight stabilizer used for MP-NPs preparation, MP-NPs significantly improved triterpenoid solubility, likely due to reduced drug particle size and the increased specific surface area (Abdollahi et al., 2024; Bali et al., 2011; Aluri et al., 2023; Wang Z et al., 2017; Sun et al., 2024; Zhang, 2018; Roos et al., 2017; Wang et al., 2020). PVP K30 stabilizes the nanocrystal system primarily through steric hindrance. Its long polymer chains adsorb onto the nanoparticle surfaces, forming a physical barrier preventing particle aggregation through steric repulsion. This spatial barrier effectively counters attractive forces (e.g., van der Waals forces) between nanocrystals, thereby preserving their reduced size and preventing agglomeration, which is crucial for maintaining the high specific surface area and the resultant increased solubility. It is also crucial for improving its dissolution rate and promoting absorption. This experiment offers a valuable reference for the exploration of various dosage forms. The toxicology and pharmacodynamics of this preparation will be explored to continue to improve nanosuspension-related research data.