The stem of Cistanche tubulosa (CT) was cut into small pieces and extracted by refluxing with water, after which the resulting filtrate was collected. The filtrate was concentrated. Ethanol was then added to the concentrate, and the supernatant was collected. This supernatant was further purified using macro-porous absorption resin and subsequently spray-dried to obtain the aqueous extract (CTE). The yield of the CTE is 10% from the dried stem of CT. This extract was produced by Sinphar Tian-Li Pharmaceutical Co., Ltd.

For this study, zebrafish embryos were treated with CTE and assigned to four treatment groups: 1) QT1: CTE, 2) QT2: Echinacoside, 3) QT3: Acteoside, and 4) QT4: Isoacteoside.

2.1.1. Zebrafish Care

Zebrafish embryos were raised at 28.5˚C, and their developmental stages were determined based on the criteria described in The Zebrafish Book. All animal procedures were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (ASIACUC) (protocol #10-12-114).

2.1.2. Microinjection of Zebrafish Embryos

Plasmid DNA (Tol2 transposon-donor plasmid) was injected into one-cell zygotes using a microinjection system consisting of a SZX9 stereomicroscope (Olympus, Tokyo, Japan) and an IM300 Microinjector (Narishige, Tokyo, Japan). The concentration of all plasmid DNA used to do the microinjection is about 500 ng/μl and the amount of all plasmid DNA injected into zebrafish embryos is about 0.2 ng. The working concentration of CTE (QT1 - 4) is 400 μM. Embryos at 48 and 72 hours post-fertilization (hpf) were observed under an Olympus IX70-FLA inverted fluorescence microscope. Images were taken using the SPOT system (Diagnostic Instruments, Sterling Heights, MI).

2.2.1. Screening for Anti-Tauopathy Effects

Truncated human tau proteins in zebrafish were tagged with green fluorescent protein (GFP). Zebrafish embryos injected with tau-GFP were treated with QT1 - QT4 at 48 and 72 hours post-fertilization. High-content immunofluorescence live imaging analysis was performed to assess anti-tauopathy effects.

2.2.2. Screening for Human-APPswe Anti-Secretase Effects

To investigate anti-secretase activity, zebrafish embryos co-expressing Bcl-2 and tau-GFP in spinal cord neurons were used. Embryos co-injected with pHuC-APPswe and pHuC-APPswe-2A-PS1 (M146V) were treated with QT1 - QT4 at 48- or 72-hours post-injection. Anti-secretase effects were evaluated using high-content direct imaging and time-lapse recording analysis.

2.2.3. Screening for Neurite Outgrowth Effects

To express GFP fusion proteins in neurons, GFP genes were driven by the zebrafish neuron-specific HuC promoter. Zebrafish neurons expressing GFP fusion proteins were treated with QT1 - QT4 for 72 hours post-fertilization (hpf), and neurite outgrowth was observed and analyzed.

A double-blind, randomized, placebo-controlled preliminary observational study was conducted to evaluate the effects of CTE on learning and memory. Eligible participants were assigned to one of three groups: Group A (600 mg CTE), Group B (300 mg CTE), or Group C (placebo).

Participants were instructed to take the assigned treatment once daily for six weeks. Healthy volunteers with Mini-Mental State Examination (MMSE) scores ≥ 24 were recruited. The study was conducted from August 8, 2008, to December 31, 2008.

The efficacy endpoint was assessed using the Clinical Memory Scale developed by the Psychological Institute of the Chinese Academy of Sciences. The scale comprises five components: 1) directed memory, 2) associative learning, 3) picture-free recall, 4) meaningless figure recollection, and 5) associative memory of portrait characteristics.

The raw scores were converted to an equivalent scale score and memory quotient. Memory ability assessments were conducted at baseline and at the end of the study.

Blood and urine samples were collected at baseline and at the end of the study for biochemical analysis, including white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin, creatinine, blood urea nitrogen (BUN), albumin, total protein, alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

The concentrations of brain-derived neurotrophic factor (BDNF) and acetylcholinesterase (AChE) were also measured.

Anti-secretase enzyme activity and neurotrophic effects of CTE in zebrafish embryos were evaluated using direct imaging and time-lapse recording, including neurite outgrowth measurements.

For the preliminary observational study, clinical Memory Scale efficacy endpoints were analyzed for participants who completed the six-week treatment. A two-sided t-test was used to assess changes from baseline, and between groups by one-way ANOVA test. P-values < 0.05 were considered statistically significant.

This study identified three key findings demonstrating that CTE exhibits antioxidative and neurotrophic properties, contributing to neuroprotection and maintaining human tau-GFP protein in its phosphorylated state, which is essential for preserving microtubule stability within neuronal axons.

3.1.1. Anti-Tauopathy Effects

Treatment with QT1 and QT3 demonstrated antioxidative and neurotrophic effects, leading to a protective outcome and maintaining human tau-GFP protein in a phosphorylated state. Truncated human tau proteins in zebrafish were tagged with green fluorescent protein (GFP) and designated as pHuC-hTau-ΔC50-GFP (Figure 1(a)). Viable GFP-positive neurons were counted in individual embryos. QT1 and QT3 effectively suppressed neurotoxicity caused by hyperphosphorylated and aggregated tau (Figure 1(b)), whereas QT2 and QT4 exhibited neurotoxic effects (Figure 1(c)).

Figure 1. Zebrafish embryos GFP-labeled neuronal cells and axons were observed at 48 and 72 hpf in embryos. (a) Truncated human tau proteins in zebrafish were tagged with green fluorescent protein (GFP) and designated as pHuC-hTau-ΔC50-GFP; (b) QT1 and QT3 effectively suppressed neurotoxicity caused by hyperphosphorylated and aggregated tau; (c) QT2 and QT4 exhibited neurotoxic effects.

3.1.2. Human-APPswe Anti-Secretase Effects

Truncated human tau proteins in zebrafish co-expressing Bcl-2 and tau-GFP in spinal cord neurons were designated as pHuC-hTau-V3-GFP-IRES-Bcl2, tagged with green fluorescent protein, and were observable. Treatment with QT1 - QT4 at 48 or 72 hours post-injection resulted in the presence of GFP-tagged neuronal cells and axons in embryos injected with pHuC-APPswe. Additionally, QT1, QT3, and QT4 effectively suppressed neurotoxicity induced by pHuC-APPswe injection. These effects are shown in Figure 2Figure 2.

3.1.3. Human Neurite Outgrowth Effects

Zebrafish embryos injected with ITR-HuC-GFP and treated with QT1, QT3, or QT4 exhibited neurite outgrowth at 72 hours post-fertilization (Figure 3Figure 3). Statistical analysis indicated that embryos treated with QT1, QT3, and QT4 had a significantly higher percentage of neuronal cells compared to controls. It is noted that QT4 promoted neurite outgrowth at 72 hours post-fertilization due to its antioxidative and neurotrophic properties.

Figure 2. Zebrafish embryos co-expressing Bcl-2 and tau-GFP in spinal cord neurons were observed at 48 and 72 hpf in embryos. (a) Truncated human tau proteins in zebrafish were tagged with green fluorescent protein (GFP) and designated as pHuC-hTau-V3-GFP-IRES-Bcl2; (b) QT1, QT3 and QT4 effectively suppressed neurotoxicity caused by injected with pHuC-APPswe; (c) QT2 exhibited neurotoxic effects.

Figure 3. Zebrafish embryos neurons expressing GFP fusion proteins were treated with QT1 - QT4 for 72 hours. (a) Zebrafish embryos injected with ITR-HuC-GFP and treated with QT1, QT3, or QT4 exhibited neurite outgrowth at 72 hours post-fertilization; (b) Statistical analysis indicated that embryos treated with QT1, QT3, and QT4 had a significantly higher percentage of neuronal cells compared to controls.

3.2.1. Demographic Analysis of Healthy Subjects

The demographic characteristics of participants who completed the study are summarized in Table 1. A total of 93 participants were enrolled, with 85 subjects (47 males and 38 females) completing the study. Participants were randomly assigned to one of three groups: Group A (n = 27), Group B (n = 32), or Group C (n = 26).

Table 1. Demographic analysis of healthy subjects.

One-way ANOVA Test.

3.2.2. Results of Memory Tests

No significant differences in memory test outcomes were observed between treatment groups. Improvements in memory tests from baseline were noted in all three groups. Pairwise comparisons of change-from-baseline scores showed statistically significant improvements (from P-value < 0.05 to P-value < 0.01) in Groups A (600 mg) and B (300 mg) compared to Group C (placebo). While Group A exhibited greater improvements than Group B, the difference was not statistically significant (P = 0.273) (Table 2).

Table 2. Memory test of study participants (N = 85).

Comparison of within group difference (change-from-baseline) is conducted by paired t-test; Pairwise comparisons of between group difference is conducted by two sample t-test. *P-value < 0.05; **P-value < 0.01.

3.2.3. Results of Safety Evaluation

Analysis of safety endpoints (Table 3) revealed no significant changes in biochemical parameters from baseline, and no treatment-related adverse reactions were observed during the study period.

Table 3. Safety endpoints of biochemical analysis in study participants (N = 85).

Comparison of within group difference in change-from-baseline is conducted by paired t-test. *P-value < 0.05; **P-value < 0.01.

3.2.4. Results of Serums BDNF and AchE Levels

Biomarker analysis (Table 4) indicated statistically significant changes-from-baseline in BDNF and AChE levels in Groups A and B, both of which received CTE treatment. These changes were also significantly greater compared to the placebo group.

Table 4. Biomarker analysis of BDNF and AChE in selected study participants.

^aComparison of within-group difference (change-from-baseline) is conducted by paired t-test; ^bPairwise comparisons of between-group difference are conducted by two-sample t-test.