Bangle Rhizome Extract Promotes Hippocampal Neurogenesis Associated with Improved Cognitive Functions: A Case Study Volume 52- Issue 5

Takuji Shirasawa^1,2*, Kazumi Hirano³, Masakazu Namihira³, Luis Carlos Aguilar Cobos⁴ and Tatefuji Tomoki⁵

• ¹Ochanomizu Health and Longevity Clinic, Japan
• ²Shirasawa Anti-Aging Medical Institute, Japan
• ³Molecular Neurophysiology Research Group, Biomedical Research Institute, The National Institute of Advanced Industrial Science and Technology (AIST), Japan
• ⁴Livant Neurorecovery Center, Mexico
• ⁵Hosoda SHC Company Ltd., Japan

Received: September 06, 2023;   Published: September 13, 2023

*Corresponding author: Takuji Shirasawa, Ochanomizu Health and Longevity Clinic, Tokyo 101-0062, Shirasawa Anti-Aging Medical Institute, Tokyo 101-0062, Japan

DOI: 10.26717/BJSTR.2023.52.008314

Abstract PDF

Bangle Rhizome Extract (BRE) promotes neurogenesis of human neural stem cells and improves spatial learning memory in ageing model mice. In the present case study, we applied BRE to a 53-year-old female patient with mild memory impairment for 6 weeks. We examined cognitive functions, P300 Electroencephalogram (EEG) signals, and MRI signals before and after the administration of BRE. Spatial memory, working memory, attention, and reaction time were improved after BRE administration in association with hippocampal neurogenesis. EEG recordings showed improved electrical abnormalities with increased neural connections and enhanced EEG responses in attention and visual spatial memory tests. MRI showed the fusion of fragmented tissues in the hippocampus after BRE administration, which represents morphological changes observed in the regenerative process of the hippocampus. This is the first clinical case report that BRE promotes neurogenesis and regeneration of the hippocampus associated with improved cognitive functions.

Keywords: Bangle Extract; Hippocampal Neurogenesis; Cognitive Function

Abbreviations: BRE: Bangle Rhizome Extract; EEG: Electroencephalogram; hfNSCs: Human Foetal Neural Stem Cells; NCI: Neurocognition Index; DG: Dentate Gyrus; SD: Standard Deviation

Bangle, Zingiber Purpureum Rosc., is a tropical ginger widely distributed in Southeast Asia. Bangle has been used as a traditional Indonesian medicine known as “Jamu” for the treatment of fever, headaches, stomach pain, rheumatism, obesity, postpartum problems, and COVID-19 infection [1,2]. Chemical analysis showed that Bangle Rhizome Extract (BRE) contains cis- and trans-3-(3,4-dimethoxyphenyl)- 4-[(E)-3,4-dimethoxystyryl] cyclohex-1-enes (c- and t-banglenes) and dimers of (E)-1-(3,4- dimethoxyphenyl) butadiene [3,4]. These chemical compounds showed neurotrophic activity in PC12 cells and protective activity against cell death caused by deprivation of serum in primary culture of mouse cortical neurons [5]. In an animal model of nondegenerative diseases, chronic treatment with BRE in senescence-accelerated prone-8 (SAMP8) mice improved spatial learning and memory deficits with enhanced hippocampal neurogenesis [6]. We recently showed that BRE induces the proliferation and differentiation of Human Foetal Neural Stem Cells (hfNSCs) by activating the canonical Wnt/ß-catenin signalling pathway as a mechanism of neuronal differentiation [7]. Safety assessment and oral toxicity tests of BRE have been performed [3]; however, clinical applications of BRE for patients with cognitive decline have not been published to date. In this study, we present for the first time a human case study in which BRE administration induced neurogenesis and regeneration of the hippocampus with improved cognitive functions.

Materials

Bangle Rhizome Extract powder (Hosoda SHC Co., Fukui, Japan) contained trans- and cis-3-(3′,4′-dimethoxyphenyl)-4-[(E)-3”,4”-dimethoxystyryl] cyclohex-1-ene (phenylbutenoid dimers; 5.0%) and was composed of 20.2% Bangle extract, 8.5% emulsifier, and 71.3% dextrin. Bangle Rhizome Extract (BRE) tablets (200 mg/tablet) containing 85 mg/tablet of BRE powder (22.7 mg/tablet of BRE containing 5 mg/tablet of c- and t-banglenes), sucrose fatty acid ester, and dextrin were made from powder, pregelatinized starch (Asahi Kasei Chemicals, Tokyo, Japan), Xantan gum (Ina Food Industry, Nagano, Japan), silicon dioxide (CDSL, Japan, Tokyo), and shellac (Gifu Shellac Manufacturing, Gifu, Japan) [3].

Cognitive Evaluation

The Cognitrax standard package was used to examine neurocognitive functions [8]. The patient performed 10 tasks displayed on a personal computer: Verbal Memory, Visual Memory, Finger Tapping, Symbol Digit Coding, Stroop Test, Shifting Attention, Continuous Performance, Perception of Emotions, Non-Verbal Reasoning, and 4-part Continuous Performance Test. Based on the scores on the 10 tasks, a composite Neurocognition Index (NCI) and 15 domain scores (composite memory, verbal memory, visual memory, psychomotor speed, reaction time, processing speed, motor speed, composite attention, sustained attention, simple attention, cognitive flexibility, executive function, social acuity, reasoning, and working memory) were calculated. Individual scores were standardized by setting the mean score to 100 and the Standard Deviation (SD) to 15; therefore, the scores in participants can be compared to those in healthy people without investigating healthy people [8]. A higher score indicates better neurocognitive function.

Electrophysiological Study

NuAmps EEG/ERP Amplifier (NeuroScanTM, https://compumedicsneuroscan. com/products-overview/) was used to collect EEG data
across 20 channels during five paradigms:
1. A resting state paradigm in which participants sat quietly with their eyes open and then closed for 3 min each,
2. A P300 paradigm that involved an active listening task in which participants were asked to count rate deviant (1,500 Hz) tones presented amongst frequent standard (1,000 Hz) tones,
3. An attention paradigm in which participants were asked to hit the keyboard when the letter “T” appeared on the screen followed by the letter “S”,
4. A visual space paradigm in which participants were asked to answer how many flashing boxes appeared on the screen, and
5. A mental flexibility paradigm in which participants were asked to determine the sorting manner of cards using the Wisconsin Card Sorting Test. Collected EEG data were analysed by NeuroScanTM Software (SCAN version 4.3, now version up to CURRY 9, https://compumedicsneuroscan.com/ products-overview/) for P300 analysis, the visual spatial memory test, the attention test, the mental flexibility test, and coherence analysis.

Cell Culture and Immunostaining

hfNSCs were purchased from Phoenix Songs Biologicals, Inc. (PSB, Branford, CT) (Cat# 23001-003, Donor Lot CxB-3). In preparation for establishing hfNSC lines, informed consent was obtained from the donor or the donor’s next of kin by the PSB company (https:// phoenixsongsbio.com/). The cell line was derived from the human cerebral cortex of a male foetus at embryonic week 14. Culture methods for hfNSCs have been described previously [7,9,10], and the neural aspect of this cell line was confirmed. In brief, hfNSCs were cultured in N2-supplemented Dulbecco’s modified Eagle’s medium with F12 (DMEM/F12, GIBCO, Waltham, MA) containing a 0.1% B27 supplement (GIBCO), 10 ng/ml human basic fibroblast growth factor (FGF; R&D Systems Inc., Minneapolis, MN), and 20 ng/ml human epidermal growth factor (EGF; PeproTech, Inc., Rocky Hill, NJ) on culture dishes that had been precoated with poly-l-ornithine (SigmaAldrich, St. Louis, MO) and laminin (Corning, Corning, NY). A maximum of 30 cell passages were used. For neuronal differentiation, hfNSCs were placed into neurobasal medium (GIBCO) containing 2% B27 supplement (GIBCO) and 0.5 mM l-glutamine (Nacalai Tesque, Inc., Kyoto, Japan). For immunostaining, cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS, and stained with appropriate antibodies against βIII-tubulin and β-catenin. Nuclei were stained after fixation using Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan). Stained cells were visualized with a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

Molecular Illustration

The 3D chemical structure of banglene, 1beta-(3,4-dimethoxystyryl)- 2alpha-(3,4-dimethoxyphenyl)-3-cyclohexene (PubChem CID 53494256), was imported from the PubChem library (https:// pubchem.ncbi.nlm.nih.gov/docs/about). The 3D protein structures of Wnt, LRP6, Frizzled, Dvl (Dishevelled), Axin, GSK 3ß, ß-catenin, LEF, and tau were retrieved from the RCSB Protein Data Bank (RCSB PDB, https://www.rcsb.org) and then imported into UCSF Chimae ra-X (https://www.cgl. ucsf.edu/chimaerax/). The molecules are displayed with molecular surfaces coloured by amino acid hydrophobicity or electrostaticity.

A 53-year-old Japanese woman developed gradual progression of memory impairment with well-preserved language comprehension, emotional control, and spatial and temporal orientation. Her cognitive function examination on December 9, 2022, using Cognitrax revealed mild reasoning impairment (Cognitrax score = 76), while working memory, reaction time, motor speed, cognitive flexibility, executive function, spatial memory, and attention were normal with a Cognitrax Total Score of 108.0. Blood chemistry, CBC, and HbA1c analysis failed to indicate any disorder associated with memory impairment. MRI data acquired on August 3, 2021, showed no remarkable atrophy of the cerebral cortex and no vascular pathologies, while endoscopic in silico view of hippocampi showed degeneration at the upper part of the neck in the left hippocampus (Figure 1A), indicated by red and blue arrows) and fragmentation at the head and upper part of the neck in the right hippocampus (Figure 1B), indicated by violet and yellow arrows). Electrophysiological evaluation on December 13, 2022, showed that asymmetrical P300 EEG responses were detected between the right parietal lead (P3) and left parietal lead (P4) (Figure 2A) red lines). The visual spatial memory test showed hyperexcitable EEG reactions at the left frontolateral lead (F7) and right frontolateral lead (F8), suggesting that degenerative pathology in the frontal lobe caused the memory problems (Figure 3B), red lines).

Figure 1

Figure 2

The attention test also showed hyperexcitable EEG responses in both frontopolar leads (FP1 and FP2) with symmetrical reactions with a later phase peak at 750 msec (Figure 3C). The mental flexibility test using the Wisconsin Card Sorting System showed a hyperexcitable reaction with 10,000 μV2 at 850 msec at the left frontopolar lead (FP1) (Figure 3D), implying that the generation of ideas may be mildly impaired due to degenerative pathology in the frontal lobe. Overall, the degenerative pathology in the hippocampus and frontal lobe, partly in the parietal lobe, may contribute to the symptoms of memory impairment. Coherence analysis showed a mild decrease in the number of neural connections at left frontolateral (F7), left temporal (T3 and T5), left central (C3), left occipital (O1), right frontolateral (F8), right temporal (T4 and T6), and right occipital (O2) leads (Figures 4A & 4C). We therapeutically applied 6 tablets of Bangle Rhizome Extract (BRE) every day for 12 weeks and evaluated cognitive function at 6 weeks and 12 weeks as well as electrophysiological evaluation at 12 weeks, as shown in (Figure 5). On February 3, 2023, 6 weeks after administration of BRE, cognitive function tests showed improvement in reasoning (76 -> 90) and cognitive enhancement in working memory (98 -> 118), spatial memory (90 -> 116), reaction time (102 -> 111), and attention (92 -> 107) with an improvement in Cognitrax Total Scores (108.0 -> 111.0) (Figure 5).

Figure 3

Figure 4

Figure 5

On March 31, 2023, 12 weeks after administration of BRE, a second cognitive function evaluation showed cognitive recovery for motor speed (109 -> 114) and maintenance of the cognitive enhancement for working memory (118 -> 118), spatial memory (116 -> 116), reaction time (111 -> 112), attention (107 -> 107), and reasoning (90 -> 90), while cognitive functions declined for cognitive flexibility (104 -> 95) and executive function (105 -> 95) (Figure 5). On July 25, 2023, after the washout period of 4 months without BRE, cognitive function evaluation showed further cognitive enhancement for working memory (118 -> 121) and reasoning (90 -> 104), while cognitive declines were observed for spatial memory (116 -> 90), attention (107 -> 77), motor speed (114 -> 105) and reaction time (112 ->107), with a decline in the Cognitrax Total Score (108.0 -> 103.0) (Figure 5). These data suggested that continuation of BRE administration may be necessary for the maintenance of improved cognitive functions such as attention, spatial memory, reaction time, and motor speed. We performed MRI on July 20, 2023, which showed the regeneration of damaged tissues in the medial part of the neck region in the left hippocampus (Figure 1C), red and blue arrows). Another endoscopic in silico view of the right hippocampus showed the fusion of fragmented hippocampal tissue (violet arrow) as well as the regeneration of fragmented hippocampal tissue (yellow arrow) (Figure 1D). This is the first report to show that the medial parts of the hippocampal neck and head degrade into fragments during the degenerative process while the fragmented tissues fuse to the lateral part of the hippocampus (defragmentation), as shown in (Figure 1D) (violet arrow, fused; yellow arrow, defragmentation) in the regenerative process. Anatomically in the anterior part of the neck portion in the hippocampus (Figures 2A & 2C), the Dentate Gyrus (DG) is located in the medial part of the neck as well as inside of the neck (Figures 2A & 2C) red arrows). Since granular cells in the DG are more vulnerable than pyramidal cells in the CA1 and CA3 regions in the degenerative process [11] or multiple sclerosis [12], the loss of granular cells in the DG may result in fragmentation and loss of the medial part of the hippocampal neck, as shown in (Figures 2B & 2D). We re-evaluated the patients’ EEG signals on March 24, 2023, which showed symmetrical P300 with higher voltages in both parietal leads after BRE administration (P3 and P4) (Figure 3A). The visual spatial memory test showed that the hyperexcitable EEG reaction observed before BRE administration was suppressed in both frontolateral leads (F7 and F8), while enhanced EEG reactions were observed in both frontopolar leads after BRE administration (FP1 and FP2) (Figure 3B), which was compatible with an improved spatial memory score with Cognitrax (Figure 5).

The attention test showed that the hyperexcitability observed before BRE administration was significantly suppressed after BRE administration (Figure 3C), suggesting that the regeneration of GABAergic interneurons suppresses the glutamatergic excitatory neurons evoked by the attention test as described previously [13,14]. The mental flexibility test also showed that the hyperexcitable reactions observed before BRE administration significantly suppressed the peak voltage of 4,500 μV2 (Figure 3D) after BRE administration, which was compatible with an improved reasoning score with Cognitrax (Figure 5). Coherence analysis showed that the number of neural connections was more enhanced after BRE administration at the left temporal (T3 and T5), left central (C3), right frontolateral (F8), right temporal (T4 and T6), and right occipital (O2) leads (Figures 4B & 4D) than the neuronal connection before BRE administration (Figures 4A & 4C), suggesting that BRE facilitated neuronal connections and synapse formation in the cerebral cortex.

Bangle, Indonesian ginger (Zingibar Purpureum), is a tropical ginger widely distributed in Southeast Asia. A previous in vitro study using human foetal neural stem cells (hfNSCs) showed that Bangle Rhizome Extract (BRE) promoted neuronal differentiation from immature neurons and accelerated neurite outgrowth [7]. As shown in (Figure 6A), hfNSCs were treated with 10 ng/mL BRE and stained with anti-ßIII-tubulin antibody 7 days after induction of differentiation. The results showed that BRE induced the differentiation of hfNSCs in vitro compared to stem cells without BRE treatment. Furthermore, BRE induced the accumulation of ß-catenin in the nuclei of hfNSCs (Figure 6B) [7], suggesting that BRE activated the Wnt/ß-catenin pathway and induced gene expression for neurogenesis [7]. A previous study suggested that the Wnt/β-catenin signalling pathway plays an important role in neurogenesis and AD pathology [15,16]. As illustrated in (Figure 7), in the Wnt-off state (blue arrows), the GSK 3ß destruction complex (GSK3ß, Axin, Dvl, and other molecules) phosphorylates ß-catenin, which is subjected to the degradation pathway by the ubiquitin‒proteasome system. GSK 3ß also phosphorylates tau at specific Ser and Thr residues that have been reported to be phosphorylated in Alzheimer’s disease [17], suggesting that activation of canonical Wnt/ß-catenin signalling may delay the progression of AD pathology (Figure 7).

Figure 6

Figure 7

In the Wnt-on state (red arrows), active Wnt ligands interact with the Frizzled receptors and the LRP5/6 coreceptor. Phosphorylation of LRP5/6 by GSK 3ß recruits Dvl and Axin to the receptor complex, which results in inhibition of the GSK 3ß destruction complex (Figure 7). In the Wnt-on state, the inhibition of ß-catenin phosphorylation stabilizes ß-catenin in the cytoplasm, which facilitates the translocation of ß-catenin into the nucleus and its association with the LEF transcriptional complex, resulting in the upregulation of the expression of genes related to neurogenesis, such as TBR2, DCX, DLX2, N-MYC, and EGFR, as previously described [7]. A previous study also suggested that BRE targets upstream molecules of the Wnt signalling pathway, such as ligands and receptors, including Frizzled (Figure 7), based on inhibitor experiments with XAV939, an inhibitor of the tankyrase (NKS) enzyme, or TWR-1-endo, an inhibitor that stabilizes the ß-catenin degradation complex [7]. It is thus speculated that BRE activates the Wnt/β-catenin signalling pathway to induce neurogenesis of neuronal stem cells in the hippocampus and cerebral cortex but also inhibits the progression of AD pathology (Figure 7). An MRI study showed the fusion of fragmented tissues in the medial part of the head and neck of the hippocampus (Figure 1D), suggesting that the neurogenesis of granule cells in the dentate gyrus of the hippocampus results in the morphological regeneration of the hippocampus with neuronal connections between granular cells in the DG and pyramidal cells in CA3 in the hippocampus (Figures 1 & 2).

Electrophysiological studies suggested that BRE induced the neurogenesis of GABAergic interneurons to suppress the hyperexcitability observed in the attention test and mental flexibility test (Figures 3C & 3D) and the neurogenesis of glutamatergic excitatory neurons, as shown in the visual spatial memory test (Figure 3B). The data suggested that BRE stimulated not only neuronal stem cells in the hippocampus but also neuronal stem cells residing in the cerebral cortex, as previously described [14,18-20].

In the present case study, we applied Bangle Rhizome Extract (BRE) to a 53-year-old female patient with mild memory impairment for 6 weeks. Spatial memory, working memory, attention, and reaction time were improved after BRE administration in association with hippocampal neurogenesis. MRI showed the fusion of fragmented tissues in the hippocampus after BRE administration, which represents the morphological changes observed in the regenerative process of the hippocampus. This is the first clinical case report that BRE promotes neurogenesis and regeneration of the hippocampus associated with improved cognitive functions.

This work was supported by a donation from HOSODA SHC Co., Ltd.

The authors would like to thank Ms. Sayuri Sato, Ms. Masami Fukuda, Ms. Fernanda Diaz, and Dr Itzel Aguilar for the preparation of this manuscript.

Written informed consent was obtained from the patient.

The authors have no conflicts of interest.

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