Comparative pharmacokinetics and hippocampal distribution of six major bioactive alkaloids in normal and Alzheimer’s disease rats after oral administration of Menispermi Rhizoma extract

2.1. Chemicals, reagents, and materials

MR was purchased from Anguo Yuntian Traditional Chinese Medicine Co., Ltd. (Anguo, Hebei, China) and identified as the dried rhizome of Menispermum dauricum DC. by Guohui Li (School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine). This product is an unprocessed crude herb. Dauricine (batch No. 6113, purity ≥ 98%) was purchased from Shanghai Standard Technology Co., Ltd. (Shanghai, China). Verapamil hydrochloride (batch No. K1629079, purity ≥ 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The other alkaloids, daurisoline, dauricicoline, acutumidine, dauricoside and 6-O-demethylmenisporphine, with purity ≥ 98%, were prepared and characterized in the authors’ laboratory. The chemical structures of all alkaloids are shown in Figure 1. Methanol and acetonitrile of LC-MS grade were purchased from Fisher Scientific (Pittsburgh, PA, USA), and formic acid (LC-MS grade) was purchased from Sigma-Aldrich (Missouri, USA). Distilled water was obtained from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China).

Close

Figure 1. The chemical structures of (a-f) the 6 alkaloids and (g) the internal standard (IS).

Export to PPT

2.2. Animals and establishment of the AD model

Sprague-Dawley rats (180 ± 20 g) were purchased from SiPeiFu (Beijing) Biotechnology Co., Ltd. (Beijing, China) and kept in SPF-grade conditions (a natural light-dark cycle at a temperature (25 ± 2 ℃) and 50 ± 10% relative humidity). All animals had free access to water and food until 12 h prior to the experiment. The experimental protocols were approved by the animal ethics committee of Tianjin University of Traditional Chinese Medicine (approval No. TCM-LAEC2020085). In this study, a total of 72 rats were employed for four experimental objectives: histopathology (n = 6), identification of absorbed components (n = 18), comparative pharmacokinetics (n = 12) and hippocampal distribution (n = 36). After acclimatization for 7 days, the rats were randomly assigned to a sham-operated group (n = 33) and an AD model group (n = 39). The rats in the AD model group were intraperitoneally injected with D-galactose (100 mg/kg/day) for 10 weeks, whereas the sham-operated rats were injected with an equivalent volume of physiological saline. On the first day of the seventh week, the rats in AD model group were injected with D-galactose, followed by bilateral hippocampal infusion of 5 μL Aβ_25-35 (1 mg/mL). By contrast, the rats in the sham-operated group received an equal volume of physiological saline via the same route. Morris water maze test and hematoxylin & eosin (H&E) staining were applied to evaluate whether the AD model was established successfully.

2.3. Morris water maze test and hematoxylin & eosin staining

Behavioral experiments and histopathological analysis were carried out to evaluate the AD model. The Morris water maze was used to evaluate spatial learning and memory abilities in AD rats through place navigation and probe trials. The brain tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 μm sections, and subjected to H&E staining for histopathological analysis. Hippocampal morphology was observed under an optical microscope (Nikon Digital Sight DS-Fi2, Japan).

2.4. Preparation of MR extract

500 g of powdered MR sample was subjected to reflux extraction twice with 95% ethanol (1:20, w/v) and twice with 75% ethanol (1:20, w/v) for 1 h each time. The filtrates were combined and concentrated to obtain the ethanol extract. The content of six active alkaloids in the MR extract was determined by UPLC-MS/MS. The analysis showed that MR extract contained dauricine, daurisoline, dauricicoline, acutumidine, dauricoside and 6-O-demethylmenisporphine at concentrations of 62.79, 85.69, 2.22, 5.10, 10.15 and 0.59 mg/g, respectively. An appropriate amount of MR extract was dispersed in a 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution for oral administration.

2.5. Instruments and analytical conditions

2.6. Preparation of standard solutions

Stock solutions of six analytes were prepared by accurately weighing each compound, dissolving the compound in a small amount of dimethyl sulfoxide, and then diluting with methanol. Subsequently, appropriate volumes of these stock solutions were mixed and further diluted with methanol to prepare a mixed standard solution containing dauricine (500 ng/mL), daurisoline (1000 ng/mL), dauricicoline (250 ng/mL), acutumidine (2500 ng/mL), dauricoside (250 ng/mL), and 6-O-demethylmenisporphine (2000 ng/mL). Working solutions were prepared by serial dilution of this standard solution with methanol. Calibration standards were obtained by adding appropriate amounts of the standard to the blank matrix. For the internal standard (IS), verapamil was dissolved in methanol to prepare solutions at 100 ng/mL and 20 ng/mL.

2.7. Method validation

The method was validated for specificity, linearity, precision, accuracy, recovery, stability and matrix effect in accordance with the US Food and Drug Administration guidance for validation of bioanalytical methods, following the procedures of the previous study [20].

2.8. Qualitative identification of absorbed components of MR in plasma and brain

Eighteen rats were randomly divided into two groups: a sham-operated group (n = 6) and an AD model group (n = 12). One week after successful model establishment, the rats in the AD model group received MR extract (2.5 g/kg/day) orally for four consecutive days, whereas those in the sham-operated group were given an equivalent amount of 0.5% CMC-Na solution. To identify the bioactive components of MR absorbed in blood, blood samples were collected from the orbital venous plexus of AD model rats at 0.25, 0.75, 1, 3, 5, 7, 8, 10, 12, and 24 h after oral administration. After centrifugation at 15000 rpm for 10 min, plasma was collected and stored at -80°C until analysis. To identify the bioactive components of MR absorbed in the brain, six rats were euthanized at 1 h after the last administration to obtain whole brain tissue samples. Brain tissues were rapidly frozen in liquid nitrogen and stored at -80°C until analysis. Blank plasma and brain tissue samples were obtained from the rats in the sham-operated group.

The plasma sample processing protocol was as follows: Plasma samples from six rats were collected at each time point. After thorough mixing, 1.5 mL of plasma was deproteinized with 6 mL of acetonitrile and vortexed for 3 min. After centrifugation at 14000 rpm for 10 min, the supernatant was dried under nitrogen gas, and redissolved in 200 μL of mobile phase (acetonitrile:water = 1:1, v/v). Following another centrifugation cycle (14000 rpm, 10 min), the supernatant was collected for UPLC-Q-TOF-MS/MS analysis.

The brain tissue processing protocol was as follows: The frozen brain tissues were homogenized with 50% methanol (1:4, w/v). 2.5 mL aliquot of the resulting homogenate was mixed with 7 mL of acetonitrile, then vortexed for 3 min. Following the centrifugation (14000 rpm, 10 min, 4°C), the supernatant was dried under nitrogen gas, and redissolved in 200 μL of mobile phase (acetonitrile:water = 1:1, v/v). After centrifugation (14000 rpm, 10 min), the supernatant was analyzed by UPLC-Q-TOF-MS/MS.

2.9. Comparative pharmacokinetic experiment of MR

Six rats from each group were orally administered with MR extract at 2.5 g/kg/day. Blood samples (250 μL) were collected from the orbital venous plexus after dosing at designated time points (0 (pre-dosing), 0.083, 0.133, 0.167, 0.333, 0.75, 1.0, 3.0, 5.0, 7.0, 8.0, 12.0, 24.0, 36.0, 48.0, and 60.0 h), and then transferred into heparin sodium-coated tubes. The samples were immediately centrifuged at 15000 rpm for 10 min (4°C). The supernatant was isolated and preserved at -80°C until analysis.

The plasma sample processing procedure for pharmacokinetic studies was as follows: An aliquot of 20 μL IS solution (100 ng/mL) was first transferred into the tubes and dried under gentle nitrogen at 37°C. Subsequently, 100 µL of rat plasma was added to the tubes and vortex-mixed for 1 min. 400 µL of acetonitrile was added and vortex-mixed for 2 min. After centrifugation at 15000 rpm for 10 min (4°C), 500 µL of supernatant was transferred, dried under nitrogen gas, and redissolved in 50 µL mobile phase (acetonitrile:water = 1:1, v/v). After centrifugation (15000 rpm, 10 min), the supernatant was directly injected into the UPLC-MS/MS system for analysis.

2.10. The distribution of six alkaloids in hippocampal tissue

The rats in sham-operated and AD model groups were orally administered with MR extract (2.5 g/kg/day). Then, they were anesthetized and subjected to craniotomy at designated time points (0.25, 0.75, 3, 10, and 24 h) after administration. Brain tissues were carefully harvested, followed by precisely separating the hippocampus regions. All hippocampus samples were immediately preserved in liquid nitrogen and stored at -80°C until analysis.

The hippocampus sample processing procedure for distribution studies was as follows: 40 µL of IS solution (20 ng/mL) was first transferred to a 2 mL tube and evaporated to dryness under a gentle stream of nitrogen gas at 37°C. The hippocampal tissue (0.1g) was homogenized in methanol (1:4, w/v) to obtain a homogenate solution. Subsequently, 100 µL of the homogenate was added to the tube containing IS and thoroughly mixed. Following the addition of acetonitrile (400 µL), the sample was vortex-mixed for 2 min and centrifuged at 15000 rpm for 10 min (4°C). The supernatant was dried under nitrogen gas and redissolved in 50 µL of mobile phase (acetonitrile:water = 1:1, v/v). After centrifugation (15000 rpm, 10 min), the supernatant was directly injected into the UPLC-MS/MS system for analysis.

2.11. Data analysis

The MR components absorbed in plasma and brain tissue were analyzed using MassLynx v4.1 (Waters Corporation). The pharmacokinetic parameters including the area under the plasma concentration-time curve to the last measurable plasma concentration (AUC_0-t), the area under the plasma concentration-time curve to time infinity (AUC_0-∞), maximum plasma concentration (C_max), time to reach the maximum concentration (T_max), elimination half-time (t_1/2), mean residence time from 0 to time (MRT_0-t) and mean residence time from 0 to infinity (MRT_0-∞) were calculated by non-compartmental model using Drug and Statistics 2.0 software (Chinese professional committee of mathematical pharmacology, Shanghai, China). Pharmacokinetic data were expressed as mean ± SD. Statistical analyses were performed using IBM SPSS Statistics 21.0. Independent t-tests were used for statistical analyses of the normally distributed data, and Mann-Whitney U tests for the non-normally distributed data. P < 0.05 was considered statistically significant.

3.1. Evaluation of the AD model

The results of Morris water maze tests (Figures 2a-c) demonstrated that AD model rats exhibited significantly prolonged swimming time to first arrival on the platform, reduced swimming time in the fourth quadrant and decreased number of platform crossings compared to sham-operated rats. These findings indicated that the AD model rats had significant spatial learning and memory deficits. Furthermore, H&E staining of the hippocampal CA1 region revealed that the neurons in the sham-operated group were densely packed with a clear nucleolus (Figure 2d). In contrast, the CA1 neurons in the AD model group rats exhibited irregular and sparse neuronal arrangements, indicating impaired structural integrity and functional degradation of neurons (Figure 2e). These findings validated the successful establishment of the AD rat model.

Close

Figure 2. Behavioral performance and hippocampal CA1 region H&E staining of sham-operated and AD model rats. (a) The number of times across the platform, (b) Swimming time in the fourth quadrant, (c) Swimming time of first arrival on the platform (mean ± SD, n = 8), **P < 0.01, *P < 0.05 vs sham-operated group. (d-e) H&E staining of the CA1 region (d: sham-operated group, e: AD model group). (scale bar, 100 μm).

Export to PPT

3.2. Validation of the methodology

3.2.1. Specificity

Representative chromatograms obtained from blank matrix, blank matrix spiked with 6 alkaloids at lower limits of quantitation (LLOQs)and IS, and actual sample after oral administration of MR extract are shown in Figures 3 and 4. The results demonstrated excellent specificity; no significant interference from endogenous substances or metabolites was observed at the retention time of the analytes and IS of plasma or hippocampus samples under the optimized chromatographic conditions.

Close

Figure 3. Representative MRM chromatograms of six active components and internal standard in plasma: (a) Blank plasma; (b) Blank plasma spiked with analytes at LLOQ and IS; (c) Plasma samples obtained at 3 h after oral administration of MR extract.

Export to PPT

Close

Figure 4. Representative MRM chromatograms of six active components and internal standard in hippocampus: (a) Blank hippocampus; (b) Blank hippocampus spiked with analytes at LLOQ and IS; (c) Hippocampus samples obtained at 3 h after oral administration of MR extract.

Export to PPT

3.3. Identification of absorbed components in plasma and brain tissue

UPLC-Q-TOF-MS/MS was employed to analyze the components absorbed in the plasma and brain tissue of AD model rats. The base peak ion chromatograms in positive ion mode were acquired by analyzing blank matrices (plasma and brain) and samples after MR treatment (Figures 5 and 6). By comparing the chromatograms of the drug-containing samples with their corresponding blank matrices and cross-referencing with literature-reported mass spectrometry data, the alkaloids absorbed in plasma and brain tissue were successfully identified.

Close

Figure 5. (a) The base peak ion (BPI) chromatograms of blank plasma, (b) Plasma after oral administration of MR extract in the AD model group , and (c-h) Six representative alkaloids absorbed into plasma.

Export to PPT

Close

Figure 6. (a) The base peak ion (BPI) chromatograms of blank brain, (b) Brain after oral administration of MR extract in AD model group, and (c-h) Six representative alkaloids absorbed into brain.

Export to PPT

A total of 24 compounds were identified in both rat plasma and brain tissue of AD rats, including 7 bisbenzylisoquinoline alkaloids, 4 morphinane alkaloids, 3 oxoisoaporphine alkaloids, 4 protoberberine alkaloids, 3 aporphine alkaloids, and 3 isoquinoline alkaloids. Among these components, 23 components were detected in both plasma and brain tissue. Notably, O-methyldauricine was exclusively detected in brain tissue, while N-methylcorydaldine was detected only in plasma. The mass spectrometry data of identified alkaloids are provided in Table S10. The fragmentation pathways of six representative alkaloids are illustrated in Figures S1-S6, and the characteristic fragments of different types of alkaloids are shown in Figure S7.

3.4. Comparative pharmacokinetics of six alkaloids in MR extract

To elucidate the differences in the absorption characteristics of six active alkaloids under physiological and pathological conditions, a comparative pharmacokinetic analysis was carried out. A rapid UPLC-MS/MS method was established and validated for the simultaneous quantification of six alkaloids in plasma from sham-operated and AD model rats after oral administration of MR extract. The mean plasma concentration-time curves of six alkaloids are presented in Figure 7. The relevant pharmacokinetic parameters are shown in Table 1.

Close

Figure 7. Mean plasma concentration-time curves of 6 alkaloids in sham-operated and AD rats after oral administration of MR extract (mean ± SD, n = 6).

Export to PPT

There was a significant difference between the plasma concentration-time curves of rats in the sham-operated group and those in the AD model group. In the AD model group, the C_max and t_1/2 of dauricine, acutumidine, dauricoside and 6-O-demethylmenisporphine were increased, while the T_max was shortened. Notably, the differences in pharmacokinetic characteristics of acutumidine and 6-O-demethylmenisporphine were particularly significant (P < 0.01). These results indicated that the pathological state of AD increased the duration of the drug in the body by increasing drug absorption, shortening the time to peak, and prolonging the elimination half-life. In AD model rats, the AUC_0-t values of acutumidine and 6-O-demethylmenisporphine were 2-3 times higher than those in the sham-operated group (P < 0.001), indicating a substantial increase in bioavailability under disease conditions. It is worth noting that although 6-O-demethylmenisporphine is a trace component in MR, it exhibits the highest C_max and AUC_0-t values, indicating its potential as a key active ingredient [6]. We speculate that the remarkably high absorption of this compound is mainly attributed to its low molecular weight (307.31 Da) and simple structure, and the synergistic effect of moderate lipophilicity (LogP = 2.33) and low pKa (5.82). Dauricoside was rapidly absorbed (T_max ≤ 1 h) in both groups, indicating efficient uptake into the bloodstream. In contrast, the C_max, AUC_0-t and AUC_0-∞ values of daurisoline and dauricicoline exhibited significant reduction in AD model rats, indicating impaired absorption and reduced bioavailability. The plasma concentration of dauricicoline was notably low in both groups. This is likely due to its low dosage and high pKa (11.00) value. Under physiological pH conditions, it primarily exists in ionization state. This ionization effect significantly reduces its lipophilicity, which in turn severely impedes the passive diffusion process, ultimately leading to its poor absorption efficiency. The results showed that the pharmacokinetic characteristics of six alkaloids were significantly different in pathological and normal states, highlighting the complex interaction between disease state and pharmacokinetic behavior.

3.5. The distribution of six alkaloids in the hippocampus

The distribution of six alkaloids in the hippocampus of sham-operated and AD model rats is presented in Figure 8. The study demonstrated that all alkaloids could cross the blood-brain barrier and distribute in the hippocampus. Our team’s previous research on the tissue distribution of 6-O-demethylmenisporphine also demonstrated that this compound could penetrate the blood-brain barrier (BBB) and exhibit remarkable distribution characteristics in brain tissue [20]. Except for 6-O-demethylmenisporphine, the concentrations of the other five alkaloids in the hippocampus of AD model rats were significantly reduced compared with those of the sham-operated rats. The concentrations decreased significantly at 0.75 h after administration (P < 0.05 or P < 0.01).

Close

Figure 8. Mean hippocampus concentration-time curves of 6 alkaloids in sham-operated and AD model rats after oral administration of MR extract (mean ± SD, n = 3), **P < 0.01, *P < 0.05 vs sham-operated group.

Export to PPT

3.6. The discussion of comparative pharmacokinetics and hippocampal distribution of six alkaloids

Accumulating evidence indicates that pathological conditions can markedly influence drug pharmacokinetics by modulating biological membrane permeability, transporter activity, and metabolic enzyme function, leading to substantial ADME differences between diseased and normal physiological conditions. Nevertheless, current pharmacokinetic investigations of TCMs predominantly utilize healthy animals, largely neglecting disease-state specific evaluations. This limitation underscores the urgent need for pharmacokinetic investigations conducted in disease-relevant models to provide precise guidance for clinical medication. In this context, the present work systematically compared the pharmacokinetic profiles of six alkaloids from MR in sham-operated and AD model rats, demonstrating significant pharmacokinetic alterations between physiological and pathological states.

The pathogenesis of AD involves four interconnected pathological processes: extracellular aggregation of Aβ, hyperphosphorylation of Tau protein, chronic neuroinflammation, and progressive neuron loss, all of which contribute to cognitive decline. Specifically, Aβ aggregates activate microglia and trigger an inflammatory cascade characterized by excessive release of cytokines (e.g., interleukin-6 (IL-6), tumor necrosis factor α (TNF-α)). Critically, these inflammatory mediators further upregulate β-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity, promoting APP cleavage and generating more Aβ peptides, thereby establishing a positive-feedback loop. Consequently, the excessive production and impaired clearance of neurotoxic Aβ species are recognized as pivotal initiating factors in AD. Notably, cerebral Aβ accumulation may influence pharmacokinetic parameters, as evidenced by decreased T_max and increased AUC [26]. The increased intestinal permeability has been observed in both AD mouse models and human patients [27], which may accelerate the transepithelial drug transport, facilitate faster entry into the systemic circulation and shorten T_max. In APP/PS1 mice, the expression of the key metabolic enzyme Cyp2c29 is downregulated. A reduction in enzyme activity would be expected to diminish first-pass metabolism, thereby leading to increased AUC [28]. In this study, AD model rats receiving MR extract exhibited reduced T_max and increased AUC for dauricine, acutumidine, dauricoside and 6-O-demethylmenisporphine, indicating the therapeutic potential of these four alkaloids in AD.

Plasma concentration-time profiles of dauricine, daurisoline, dauricicoline, and acutumidine exhibited double-peak absorption phenomena, which were consistent with our previous report [19]. The bimodal phenomenon may be attributed to enterohepatic circulation, reabsorption, and biotransformation processes [29]. Additionally, fluctuations in gastric pH and diverse absorption sites along the gastrointestinal tract may also contribute to the double-peak profile [30]. The kidneys are major organs for drug metabolism and excretion. Research has indicated that the kidneys of amyloid precursor protein transgenic mouse line 23 (APP23) exhibit more severe pathological damage than those of healthy controls [31]. Impaired renal function reduces drug elimination, potentially causing drug accumulation in the body. Pharmacokinetic analysis revealed that the t_1/2 and AUC_0-t of dauricine, acutumidine, dauricoside and 6-O-demethylmenisporphine were increased, indicating slower elimination and accumulation of these four alkaloids. This phenomenon may be attributed to renal injury associated with AD.

P-glycoprotein (P-gp) is an ATP-dependent transporter expressed extensively in intestinal epithelium, blood-brain barrier endothelium, renal proximal tubules, etc. [32]. P-gp plays a pivotal role in drug disposition by pumping exogenous substances out of cells. A study revealed that when fecal supernatants from AD patients were co-incubated with polarized T84 intestinal epithelial cells, P-gp expression in the experimental cells was significantly downregulated, suggesting that AD may reduce P-gp expression in intestinal epithelial cells [33]. Notably, dauricine, a prototypical P-gp substrate, undergoes P-gp-mediated efflux during intestinal absorption, which restricts its uptake into the systemic circulation [34]. Consequently, AD-related downregulation of intestinal P-gp may attenuate first-pass elimination of dauricine, thereby increasing absorption and elevating C_max and AUC.

The hippocampus, a brain region critical for learning and memory, is among the most severely affected areas during the progression of AD. To explore the impact of hippocampal damage on drug efficacy, the current research investigated the hippocampal distribution of six components in sham-operated and AD model rats. The concentrations of five alkaloids were significantly lower in the hippocampus of AD rats compared to sham-operated rats, with the exception of 6-O-demethylmenisporphine. This discrepancy is likely due to the progressive neuronal loss and cellular damage in the hippocampus of AD rats, which may hinder drug absorption [35]. Notably, the concentration of 6-O-demethylmenisporphine in the hippocampus of AD rats was markedly elevated compared to that of sham-operated rats, suggesting that it might serve as a key active component of MR for AD treatment. Additionally, the six alkaloids exhibited distinct variations in BBB permeability. Specifically, dauricine demonstrated excellent BBB permeability, primarily due to its high lipophilicity and high oral dosage. In contrast, despite sharing a similar methoxy-substituted bisbenzylisoquinoline alkaloid structure, daurisoline and dauricicoline exhibited markedly lower hippocampal concentrations. This phenomenon may be attributed to their high pKa values (10.87 for daurisoline and 11.00 for dauricicoline) and increased polarity from hydroxyl substitution. At physiological pH, high pKa values result in the molecules being predominantly ionized, which drastically reduces their effective lipophilicity and consequently impairs passive diffusion across the BBB. Dauricoside contains a strongly polar glycosyl group, which significantly enhances its water solubility while reducing its lipophilicity, ultimately resulting in extremely poor BBB permeability. Besides, according to our previous research, we hypothesize that the excellent absorption and BBB permeability of 6-O-demethylmenisporphine may be attributed to its low molecular weight and high lipophilicity (LogP = 2.33) [20].