EDM (EDM >99%) and DEDM (DEDM >99%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). Acetonitrile (HPLC grade) was got from fisher chemical (Massachusetts, United States). Non-essential amino acids solution from Sigma-Aldrich (Shanghai, China) and Hank’s balanced salt solution (HBSS), Penicillin-Streptomycin Solution (×100) and MEM were purchased from Hyclone (Massachusetts, United States). Sodium pyruvate was bought from solarbio science and technology Co., Ltd., (Beijing, China). Gibco (New York State, United States) supplied the 0.25% Trypsin-EDTA and penicillin-streptomycin solution. From Every Green Biotechnology Co., Ltd., (Zhejiang, China), FBS was bought. DMSO was obtained from Meilunbio (Dalian, China). 12-well Transwell^® plate was acquired from Corning (New York State, United States). The preparation of aqueous solutions uses double-distilled water and all additional solvents are analytical agents.

The Center of Cellular Resources at the Chinese Academy of Sciences (Shanghai, China) provided the human colon adenocarcinoma cell line Caco-2, and the experiment utilized 15–30 generations of these cells. DMEM enriched with 15% FBS, 1% non-essential amino acids, 1% Penicillin-Streptomycin Solution (×100), and 1% sodium pyruvate was utilized to cultivate cells at 37°C in a humidified atmosphere of 5% CO₂. The cells were separated from flasks when they achieved the anticipated confluence threshold (70%–80%), and they were then inoculated in 12-well plates at a density of 2–5 × 10⁵ cells/cm² in a polycarbonate Transwell^® (pore size: 0.4 µm; filter area: 1.12 cm²). In the first week, the culture media was switched every 2 days. After that, the culture medium was switched out daily until the transepithelial electrical resistance (TEER) for transport experiments reached 200 Ω/cm²; It has been demonstrated in our prior experiments that this process requires 18–21 days under the specified culture conditions (Huang et al., 2020).

The cytotoxicity of EDM and DEDM was assessed in Caco-2 cells using the aforementioned MTT test. These alkaloids were produced as solutions in DMSO and then diluted in cell culture media. Inoculating Caco-2 cells (1 × 10⁴cells/well) in 96-well plates with 200 μL of media. After additional 48 h of incubation with EDM and DEDM (final test alkaloids concentrations ranged from 0.4 to 200 μM, and DMSO was less than 0.05%), the medium was discarded. To each well, an aliquot of 190 μL freshly prepared DMEM (serum-free) having 10.0 μL MTT (5 mg/mL) was added. The medium in every well was then removed from the plate after a further 4 h at 37°C of incubation. In 100 μL of DMSO, the purple formazan product was dissolved. Using a microplate reader, the ratio of absorbance (treated to untreated cells) at 490 nm was employed to measure cell viability.

Caco-2 monolayers were pre-cultured in HBSS for 15 min at 37°C after being rinsed two times with preheated Hanks' balanced salt solution (HBSS, pH = 7.4). The donor side was supplemented with DEDM (25, 50, and 100 μM) and EDM (2.5, 5, and 10 μM), and the receiver chamber was filled with the corresponding volume of HBSS medium. For 120 min, the plate was incubated at 37°C. At 0, 30, 45, 60, 90, and 120 min, a 0.5 mL aliquot of each receiver chamber’s sample was taken, and 0.5 mL of HBSS was added to keep the volume constant. By using high-performance liquid chromatography (HPLC), the samples were examined. The sample (300 μL) taken from the receiver chamber was mixed with 300 μL of acetonitrile, and the combination was centrifuged for 10 min at 13,000 rpm. Lastly, a 100 µL aliquot of the supernatant was injected into the HPLC for examination.

Two drugs were quantitatively determined using an Agilent 1,260 HPLC apparatus (United States). A C18 column (1.8 μm, 3.0 × 150 mm; Agilent Technologies, Santa Clara, CA, United States) was used for the separation, with a column temperature of 25°C. The mobile phase comprised both A (water) and B (acetonitrile). The ratio between A and B was 70: 30 with a flow rate of 1 mL per minute. For EDM or DEDM, 20 μL samples were injected and analyzed at 290 nm or 344 nm, respectively. Acetonitrile was used to create the standard stock solutions for EDM and DEDM, respectively. By serially diluting standard stock solutions with HBSS, working solutions were made. The effective concentrations were 0.04–2.5 μg/mL for EDM and 0.23–7.5 μg/mL for DEDM, respectively. These samples were stored at 4°C before being used.

Eqs 1, 2 were used to determine the apparent permeability coefficients (Papp) and efflux ratio (ER) of marker drugs: P a p p = d Q / d t C 0 A (1) where dQ/dt is the cumulative transport rate (M/min), C₀ is the initial concentration of each marker drug, and A is the surface area of the filters. E R = P a p p B L − A P / P a p p A P − B L (2)

Data for transport experiments are presented as the mean ± SD of six independent experiments.

252 male KM mice (25 ± 2 g) were purchased from the Hunan SJA Laboratory Animal Co., Ltd., China. (China. Certificate: SYXK [Xiang] 2019-049). Each mouse was kept in a climate-controlled environment (22°C–28°C) with unrestricted access to food and water and a regular 12 h light/dark cycle. Before the experiment, they spent 2 days adjusting to their housing. All mice used by the Institute of Material Medical Integration and Transformation for Brain Disorders (IBD2020007) received human care in accordance with the standards established by the National Institutes of Health. The Chengdu University of Traditional Chinese Medicine’s research committee approved this project.

In a sterile mortar, 2-hydroxypropyl-β-cyclodextrin and EDM or DEDM were added. The mixture was then transferred to a sterile centrifuge tube after being repeatedly ground for 30 min with a tiny amount of ordinary saline added. After that, saline was added and ultrasonically processed for 1 h. In a vacuum freeze drier, the suspension was freeze-dried for 30 h. To make the injection, the freeze-dried powder was ultimately redissolved in regular saline. After acclimatization, all mice were acclimated before being randomly placed into four groups of three each: EDM (H) group, EDM (L) group, DEDM (H) group, DEDM (L) group. Food was discontinued 12 h before the trial. Mice in EDM (H) group and EDM (L) group were injected EDM at the dose of 5 mg/kg or 2.5 mg/kg, and mice in DEDM (H) group and DEDM (L) group were injected DEDM at the dose of 10 mg/kg or 5 mg/kg. The blood was drawn and then promptly placed into a heparinized tube at the time points of 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 12 h after dose (at each time point). Supernatant plasma from the collected sample was transferred into another tube and kept at −80°C until analysis after being centrifuged at 3,000 rpm for 10 min. Immediately after blood was obtained, the mice were dissected. Saline was injected into the heart until the liver turned white. Then, the organs of the heart, liver, spleen, lung, kidney, and brain were removed, weighed, and homogenized (2 mL normal saline per 1 g tissue sample, 30 Hz, 1.5 min) before being kept at - 80°C for analysis. The calibration standards, tissue samples, and plasma samples were pretreated with acetonitrile for protein precipitation. To a 100 µL portion of each of the plasma or tissue sample, 200 µL of acetonitrile with internal standard was added. To remove the denatured protein, the mixture was vortexed for 1 min and then centrifuged at 16,500 rpm for 10 min. The supernatant was finally injected into the LC-MS/MS in an aliquot of 100 µL for analysis.

The Agilent 1,260 HPLC-MS/MS system has a thermostatically controlled column compartment, an online degasser, a quadruple pump, and an auto-sampler. Agilent Eclipse Plus C8 RRHD (3.0 × 150 mm, 1.8 μm) was used to perform the liquid chromatographic separation. Using an isocratic elution, the EDM and DEDM were analyzed. Both A (0.1% formic acid) and B (acetonitrile) were contained in the mobile phase. With a flow rate of 0.3 mL/min, the ratio between A and B was 20: 80 for 4 min. The column temperature was kept at 35°C. Prior to injection, samples were transferred in an auto-sampler at a temperature of 4°C and 5 µL was the injection volume. The EDM and DEDM were quantitated by using an Agilent 1260-6460 mass spectrometer. The ion source was electrospray ionization (ESI) and adopts positive ion mode. The scan mode was multiple reaction monitoring (MRM). The operating conditions for the EDM and DEDM were 4000 V capillary voltage. The dry gas flow was 11 min per liter, and the dry gas temperature was 300°C. Ion source gases 1 (GS 1) and 2 (GS 2) were both set to 15. The EDM and DEDM were detected through MRM modes with m/z transition at 304.3→171 for EDM and 301.3→286 for DEDM (for details, see Supplementary Table S1).

Acetonitrile was used to prepare standard stock solutions for each type of EDM and DEDM individually. By diluting the standard stock solutions serially with acetonitrile, working standards for the EDM and DEDM were made. The calibration curve samples were acquired by spiking the series working standard solutions (10 μL) into a tube and evaporating the solvent, and then adding blank mouse tissue homogenate or plasma (100 μL). Thus, the effective concentrations of EDM and DEDM in tissue homogenate or plasma samples were 1.8–364 ng/mL. These samples were stored at 4°C before being used. DEDM is used as an internal standard compound when detecting EDM. Meanwhile, when DEDM is detected, EDM is used as the internal standard compound. The final concentration of the internal standard compound is 10 ng/mL. The research shows EDM and DEDM do not interconvert in vivo (Zhang Z. et al., 2018; Wang et al., 2018), so they can be used as internal standard compounds for each other.

For the Non-compartmental PK analysis, Thermo Kinetica (Version 5.0, Thermo Electron Corporation, Waltham, MA) was employed. Based on observed data, the time when the maximum tissue concentration was reached (T_max) and the maximum tissue concentration (C_max) were assessed. By using the linear trapezoidal rule, the area under the plasma-concentration-time curve was calculated from 0 h to infinity (AUC_0-∞) and from 0 h to 12 h. Additionally, the mean residence time (MRT) was computed. PK parameters are reported as the mean of seven independent experiments. Linear and non-linear regressions were analyzed in Microsoft Excel 2016^® (United States).

Drug potency is largely determined by its physicochemical qualities. SwissADME is a free online tool for assessing small molecule pharmacokinetics, drug-likeness, and medicinal chemistry friendliness (Daina et al., 2014; Daina and Zoete, 2016; Daina et al., 2017). We obtained EDM and DEDM physical representations from the SwissADME version 15 database.

Electrostatic interactions, as a key feature of intermolecular non-covalent interactions, significantly affect the permeability of compounds. Intermolecular electrostatic interactions are often described accurately by molecular electrostatic potential (MEP) surfaces. PubChem was used to get the two indole alkaloids' structures (Kim et al., 2016). Using the AM1-BBC technique, the molecules' partial charges were added in Chimera (Pettersen et al., 2004). The MEP values were calculated using the adaptive Poisson-Boltzmann solver (APBS) (Baker et al., 2001). The dielectric coefficients of the compound and solvent were set to 2 and 78.5, respectively and all other parameters were set to default values. Molecular surfaces were visualized employing Chimera (Chimera version 1.12).

Cell morphology was examined under a microscope over the course of the 21 days. Results indicated that the cell boundary is clearly defined and that the cell shape is homogeneous, compact, and flat. The experimental conditions were met by the final resistance TEER, which is larger than 500/cm², showing that the model is appropriate for transport study. The HBSS chromatogram can be compared to the matching spiked samples' chromatogram to demonstrate the method’s high selectivity. No obvious HBSS interference was seen for the two identified drugs, as shown in Supplementary Figure S1. For EDM and DEDM, the retention times were 4.067 and 5.869 min, respectively. Over the concentration range of 0.04–2.5 g/mL for EDM and 0.23–7.5 g/mL for DEDM, the calibration curves were linear (see Supplementary Table S2 for details). The lowest limit of quantification (LLOQ) for EDM was 0.03 g/mL and 0.2 g/mL for DEDM, and the signal-to-noise ratios were greater than 10. The average recoveries for the three concentrations range between 90% and 110%. Both EDM and DEDM’s intra-day and inter-day accuracy (RSD,%) were under 15.0.

For the transport studies, we used EDM (2.5, 5, and 10 μM) and DEDM (25, 50, and 100 μM) based on the findings of the non-toxic concentration trials (for more information, see Supplementary Table S3). Bidirectional tests, as shown in Figure 2, revealed transmembrane drug transport across Caco-2 cell monolayers. The P _app value of DEDM found in the current investigation was at the level of 10⁻⁵ cm/s and is attributed to compounds with high permeability. However, EDM’s P _app values were at a level that is regarded as having medium permeability (10^–6 cm/s). Comparing bidirectional transport for EDM and HEDM reveals that the permeability in the secretory direction (basolateral→apical) and the absorptive direction (apical→basolateral) were nearly equal.

By contrasting the chromatograms of blank tissue homogenates or plasma samples taken from six drug-free mice with the matching spiked tissue homogenates or plasma samples, the method’s selectivity was evaluated. Figure 3 demonstrates that neither mouse tissue homogenates nor plasma significantly influenced the retention periods of either analyte. EDM and DEDM had retention times of 2.919 and 1.814 min, respectively. EDM and DEDM calibration curves were linear for the 1.8–364 ng/mL concentration range (see Supplementary Tables S4, S5 for details). The lower limit of quantification (LLOQ) was 0.9 ng/mL with a signal/noise ratio greater than 10. The signal to noise ratio (SNR) of EDMs and DEDMs was greater than 10, and the lower limit of quantification (LLOQ) was 0.9 ng/mL. For the three concentrations, the average recovery of mouse tissue homogenates or plasma ranged from 90% to 110%. For both EDM and DEDM, the intra-day and inter-day precision (RSD,%) was under 15.0. The mean IS-normalized matrix factor of the three concentrations was over than 93.7% in plasma and tissues. The results demonstrated that this quantitative approach is capable of detecting two alkaloids quantitatively in plasma and tissues. The analyses were carried out at room temperature (20°C–24°C) in triplicate.

Following tail vein injection, the concentrations of EDM and DEDM in mice’s plasma (Figure 4) and tissue (Figure 5) as well as the primary pharmacokinetic characteristics of EDM and DEDM (Table 1) were displayed. Figure 6 depicts the variations in EDM and DEDM content in several mouse organs. These findings demonstrated that the plasma concentration of EDM and DEDM reached its maximum concentration (C_max) within 15 min and then began to exponentially decline. After reaching its peak concentration in 15 min, the distribution of DEDM in different tissues falls exponentially, and the half-life (T_1/2) and mean residence time (MRT) of EDM in different tissues are longer. Both EDM and DEDM were found in the brain, with DEDM having a 6.44 times higher drug concentration than EDM at a dose of 5 mg/kg. Both EDM and DEDM were observed to cause pulmonary enrichment, reaching maximum concentrations of 113.757 μg/g for EDM and 11.267 μg/g for DEDM at doses of 5 mg/kg. Second, the order of EDM tissue distribution concentration is kidney, brain, heart, liver, and spleen, whereas DEDM tissue distribution concentration is kidney, heart, brain, liver, and spleen. The distribution of EDM in the liver, spleen, and lung was significantly higher than that of DEDM at the same dose (5 mg/kg). In contrast to DEDM, EDM was less widely distributed in the heart, kidney, and brain.

The distribution of EDM in the spleen and kidney showed no dosage difference, indicating that the organs' propensity for saturation with EDM. The largest concentrations of EDM after intravenous injection of 2.5 mg/kg or 5 mg/kg were found in the spleen, where they were 7.473 μg/g and 8.14 μg/g, respectively. The highest concentrations in the kidney were 0.481 μg/g and 0.332 μg/g, respectively. The distribution of DEDM in the kidney (1.075 μg/g or 1.508 μg/g) and brain (0.708 μg/g or 0.694 μg/g) likewise approached saturation after injections of 5 mg/kg or 10 mg/kg DEDM into the tail vein.

Table 2 presents physicochemical details of the two indole alkaloids. The SwissAEDM database was used to generate the molecular weights, LogP values, hydrogen bond donors and acceptors, topological polar surface areas (tPSAs), and rotatable bonds reported in this table. There was essentially no difference between the alkaloids in terms of molecular weights, LogP values, hydrogen bond donors and acceptors, topological polar surface areas (tPSAs), and rotatable bonds. The calculation of atomic charge to determine molecular electrostatic potential (MEP) is an easy way to describe molecule polarity. Figure 1B displays the molecular electrostatic potential (MEP) surfaces of EDM and DEDM, and Supplementary Table S6 displays the atomic charges. The outcomes demonstrated that the two alkaloids have identical atomic charges.

The bioavailability radar map displays the computed physicochemical data of compounds in the context of the oral drug-like property space. Figure 1C displays the bioavailability radar for EDM and DEDM. With the exception of saturation, EDM and DEDM’s lipophilicity, size, polarity, and flexibility were all within the ideal range and did not differ noticeably. By utilizing the carbon percentage in sp³ hybridization, saturation is a technique for describing the three-dimensional structure of a molecule. As a result, we looked at the stereostructure of two alkaloids and discovered that rings D and E and rings A, B, and C are on opposite planes, creating a dihedral angle (Figures 1A, D).