Observation of Inhalation of CXMCI-01 Essential Oil on Patients With Mild Cognitive Impairment

\< Trial Description and Rationale for Intervention \>

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Lewy body dementia (LBD), and frontotemporal dementia (FTD) are major causes of cognitive decline, with AD being the most prevalent. Dementia is characterized by cognitive impairment, memory deficits, and neuropsychiatric symptoms, leading to loss of independence and increased caregiver burden. Current pharmacological treatments can only slow disease progression, with no curative therapy available.

The pathogenesis of neurodegeneration involves multiple mechanisms, including amyloid-beta (Aβ) aggregation, tau protein hyperphosphorylation, neuroinflammation, oxidative stress, and cholinergic dysfunction. Neuroinflammation, primarily mediated by microglia and astrocytes, plays a central role in neuronal damage. Dysregulated microglia contribute to synaptic dysfunction, while pro-inflammatory astrocytes promote neuronal apoptosis through the NF-κB pathway. Oxidative stress exacerbates neurodegeneration by inducing mitochondrial dysfunction and increasing reactive oxygen species (ROS), which further promote Aβ accumulation and tau pathology.

Mild cognitive impairment (MCI) represents an intermediate stage between normal cognition and dementia. Approximately 25 percent of individuals with MCI progress to dementia within two years, while others may recover cognitive function. Current interventions, including cholinesterase inhibitors, dietary supplements, and exercise, have shown inconsistent results. Given the multifactorial nature of MCI and the potential for cognitive recovery, exploring alternative neuroprotective therapies is essential.

Olfactory dysfunction is an early biomarker of neurodegeneration, as the olfactory nerve directly connects to the limbic system, which regulates memory and emotion. Aromatherapy, the therapeutic use of essential oils, has been investigated for its effects on cognitive function. Studies on cellular and animal models have demonstrated that essential oils exert neuroprotective effects through multiple mechanisms, including reducing Aβ-induced neurotoxicity, modulating microglia-mediated neuroinflammation, regulating glutamate and GABA neurotransmission, and inhibiting tau hyperphosphorylation via glycogen synthase kinase-3β (GSK3β) suppression. Essential oils also activate cAMP response element-binding protein (CREB) signaling, enhance antioxidant defense by increasing superoxide dismutase (SOD) levels, reduce pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and modulate brain-derived neurotrophic factor (BDNF) expression. Additionally, they prevent neuronal apoptosis and mitochondrial dysfunction, inhibit acetylcholinesterase (AChE) activity, and enhance synaptic plasticity and neurotransmitter balance.

Several essential oil constituents, including 1,8-cineole, linalool, limonene, thymol, alpha-humulene, cis-carveol, alpha-pinene, beta-pinene, rosmarinic acid, and zingerone, have demonstrated neuroprotective properties. These compounds may contribute to cognitive enhancement by reducing neuroinflammation, oxidative stress, and synaptic dysfunction.

Based on this hypothesis, the investigators formulated a natural essential oil blend rich in 1,8-cineole, linalool, limonene, and other bioactive compounds, and applied it via inhalation as an intervention. Using the concept of olfactory training and targeting the limbic system, the study aims to evaluate whether essential oil inhalation can promote improvements in cognition, memory, sleep, mood, and quality of life in individuals with MCI. The effects will be assessed through validated clinical scales and by measuring urine and serum biomarkers related to neuroinflammation. Additionally, changes in meridian energy and autonomic nervous system activity will be monitored. The goal is to provide a convenient and supportive complementary therapy for individuals with MCI, offering clinicians an evidence-based option for early intervention in cognitive decline.

\< Study Procedures and Design Rationale\>

This study is a randomized, double-blind, parallel-group clinical trial designed to evaluate the effects of CXMCI-01 essential oil inhalation on cognitive function in individuals with mild cognitive impairment (MCI). A total of 100 participants will be enrolled and randomly allocated in a 1:1 ratio to either the experimental group or the control group.

Randomization will be conducted using a computer-generated sequence, and allocation will be concealed using opaque, sealed envelopes. Each participant will receive a sequential identification number. The randomization process and distribution of the essential oil formulations will be managed by a third party who is independent of the study procedures to ensure blinding.

Participants will be recruited through neurology outpatient clinics at China Medical University Hospital, Chung Shan Medical University Hospital, and Everan Hospital. Those who meet preliminary eligibility will undergo final screening and informed consent at Everan Hospital.

The study consists of three scheduled visits:

* Visit 1 (V1): Baseline assessment prior to intervention. * Visit 2 (V2): Post-intervention assessment after 28 days of essential oil inhalation. A visit window of + 5 days from the scheduled date is acceptable. * Visit 3 (V3): Follow-up assessment conducted 28 days after the completion of the observation period. A visit window of ±5 days is also acceptable.

The essential oil intervention includes two types of blended formulations, each prepared in two concentrations (100% and 0.1%):

* CXMCI-01-M: A blend containing lemon, rosemary (1,8-cineole type), rose geranium, and sweet marjoram essential oils. * CXMCI-01-N: A blend containing frankincense, true lavender, linalool, and ylang-ylang essential oils. * The control group are diluted to 0.1% concentration using a neutral carrier oil.

Participants will perform inhalation twice daily for 28 consecutive days. And use twice daily. The essential oil intervention was designed with consideration of the body's natural circadian rhythm. The autonomic nervous system exhibits daily fluctuations, with sympathetic activity typically dominant in the morning and parasympathetic activity more prominent at night. Cortisol levels also follow a diurnal pattern, peaking in the early morning and gradually declining throughout the day. To align with this physiological rhythm, the morning formulation (CXMCI-01-M) was selected to support alertness and sympathetic activation, while the evening formulation (CXMCI-01-N) was intended to promote relaxation and enhance parasympathetic activity. This time-specific approach aims to optimize the neurophysiological effects of essential oil inhalation, supporting both cognitive performance and overall well-being.

* In the morning, 2 drops of the assigned essential oil (intervention group with 100% formula, placebo group with 0.1% formula) will be applied to a cotton pad. Participants will first inhale the aroma near the nose for 5 minutes before placing the cotton pad in an aroma necklace, which will be worn for an additional 60 minutes. * The same procedure will be repeated at night with a different formulation.

The experimental group will receive the 100% CXMCI-01 formulations (CXMCI-01-M in the morning and CXMCI-01-N at night), while the control group will receive the 0.1% diluted versions of the same formulas.

To ensure consistency, participants will be instructed to avoid interfering factors during inhalation, such as eating, drinking strong-smelling beverages, smoking, or being in scented environments. Concurrent use of other essential oils, perfumes, or cognitive-enhancing treatments will be restricted throughout the study period.

This structured inhalation protocol, utilizing specific essential oil compositions with known neuroprotective properties, is designed to target neuroinflammatory and oxidative mechanisms through the olfactory-limbic pathway. The study aims to explore whether this intervention can support cognitive, emotional, and physiological well-being in patients with MCI.

Throughout the study period, participants or their caregivers will be asked to maintain a daily log, recording aromatherapy usage, sleep patterns (including bedtime, time to fall asleep, and wake-up time), physical activity (type and duration), and dietary habits. Specific attention will be given to the intake of refined sweets, sugary beverages, and fried or grilled foods. Participants will also document any acute medical events, such as hospital visits, medication use, and symptoms of upper respiratory infections. Any adverse reactions potentially related to essential oil use, such as dizziness, palpitations, or asthma-like symptoms, will be reported in the log.

Following the post-intervention assessment, participants will continue to maintain their daily tracking logs as previously instructed. A follow-up evaluation will be conducted within 28 ± 5 days after the completion of the intervention to examine the sustained effects of the essential oil inhalation.

The follow-up assessments will include a combination of clinical, cognitive, psychological, and physiological evaluations. Cognitive assessments will include the Contextual Memory Test (CMT), a standardized tool for assessing episodic memory in real-life contexts; the Montreal Cognitive Assessment (MoCA), commonly used to screen for mild cognitive impairment; and the Taiwan Odd-Even Number Sequencing Test (TOENST), which evaluates working memory and processing speed.

Psychological and quality of life assessments will include the Pittsburgh Sleep Quality Index (PSQI), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), and the 36-Item Short Form Health Survey (SF-36), which collectively assess sleep quality, emotional well-being, and overall health-related quality of life.

To explore the potential biological effects of the essential oil intervention, blood samples will be collected for biomarker analysis. These may include markers related to amyloid and tau protein pathways, such as the Aβ42/40 ratio, total tau protein, and phosphorylated tau protein-217 (p-tau 217), which are commonly associated with neurodegenerative processes and Alzheimer's disease pathology. Including these biomarkers will allow for an exploratory evaluation of possible changes in biological mechanisms relevant to cognitive decline and mild cognitive impairment.

In addition to blood-based markers, urinary biomarkers related to neuroinflammation will also be assessed at baseline and post-intervention. These may include lipid peroxidation (LPO) and 8-hydroxy-2-deoxyguanosine (8-OHdG), quinolinate, kynurenine, kynurenate, and picolinate. These biomarkers are involved in oxidative stress, mitochondrial function, and neuroimmune activity, and may provide further insights into the systemic biological response to the intervention.

Physiological assessments will include non-invasive testing using the Meridian Energy Analysis Device (M.E.A.D.) to evaluate meridian energy distribution, and heart rate variability (HRV) testing to assess autonomic nervous system function. These comprehensive follow-up evaluations aim to assess the longer-term impact of CXMCI-01 essential oil inhalation on cognitive function, emotional status, sleep quality, biological markers of neuroinflammation and neurodegeneration, and systemic physiological regulation.

To minimize variability, participants will follow dietary and lifestyle restrictions before each testing session. This includes avoiding excessive oily food and refined carbohydrates for three days before testing and refraining from alcohol or caffeinated beverages the day before. Morning urine samples will be collected on scheduled days, and female participants will be asked to avoid urine collection during menstruation.

This comprehensive assessment protocol aims to evaluate no

• Montreal Cognitive Assessment(MoCA) [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Taiwan Odd-Even Number Sequencing Test (TOENST) [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Beck Depression Inventory (BDI) [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Beck Anxiety Inventory (BAI) [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Pittsburgh Sleep Quality Index (PSQI) [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• 36-Item Short Form Health Survey (SF-36) [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Plasma biomarkers related to amyloid and tau pathology [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Urinary biomarkers related to oxidative stress and neuroinflammation [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2)]
• Meridian energy assessment using M.E.A.D device [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]
• Heart rate variability (HRV) assessment [Time frame: Baseline (V1), Post-Intervention (Day 28) (V2), and Follow-up (Day 56) (V3).]