**Background:** Alzheimer's disease (AD) is a neurodegenerative condition characterized by neuronal loss, cognitive decline, and pathological features including amyloid beta (Aβ) plaque deposition and neuroinflammation. Aβ peptides reduce neural stem cell survival, proliferation, and neurogenic differentiation. Kaempferia parviflora (KP), a plant in the Zingiberaceae family, has demonstrated anti-inflammatory, antioxidant, and anti-AD properties in previous studies, including inhibition of acetylcholinesterase and butyrylcholinesterase. This study hypothesized that KP extracts could suppress Aβ-mediated neuroinflammation and promote neuronal differentiation as a novel mechanism for AD prevention.
**Methods:** KP rhizomes were extracted with 95% ethanol to obtain crude extract (KP1), then partitioned with hexane (KP2), chloroform (KP3), ethyl acetate (KP4), and residue (KP5). HPLC analysis quantified phenolic compounds and methoxyflavone derivatives (DMF, PMF, TMF). Three cell types were used: NE-4C neural stem cells, BV-2 microglial cells, and NE-4C-derived differentiated neurons. Experiments were conducted in monoculture and co-culture systems. Cytotoxicity was assessed using XTT reduction and ATP assays. Anti-inflammatory effects were evaluated by measuring IL-6 (ELISA), nitrite (Griess reagent), and iNOS mRNA (RT-qPCR). Oxidative stress was measured using DCF fluorescence. Neurogenesis was assessed by βIII-tubulin and MAP-2 mRNA expression via RT-qPCR. Aβ42 was used at 5 µM for cytotoxicity studies and 1 µM for inflammation studies; a subtoxic dose of 0.25 µM was used for neurogenesis experiments. Statistical analysis used one-way ANOVA with Tukey's test (p ≤ 0.05).
**Key Results:** KP extraction yielded KP1 (4.67%), KP2 (18.6%), KP3 (78.0%), KP4 (0.49%), and KP5 (0.67%). TMF was the most abundant flavone derivative (KP1: 517.19 ± 2.10 mg/g; KP3: 649.54 ± 6.67 mg/g). Aβ42 at ≥5 µM showed significant cytotoxicity to all cell types. KP1, KP2, and KP3 at 2–8 µg/mL effectively normalized Aβ42-mediated neurotoxicity in both monoculture and co-culture. In BV-2 monoculture, KP1, KP2, and KP3 significantly reduced IL-6 levels (4 and 8 µg/mL), nitrite (8 µg/mL), and DCF fluorescence (KP1 and KP3 from 1–8 µg/mL; KP2 at 4 and 8 µg/mL). In co-culture between differentiated NE-4C and BV-2 cells, KP1 and KP2 at 4–8 µg/mL and KP3 at 8 µg/mL reduced IL-6 and iNOS mRNA and ROS in BV-2 cells. KP1 and KP2 (2, 4, and 8 µg/mL) and KP3 (4 and 8 µg/mL) prevented neuronal death and oxidative stress in differentiated NE-4C cells. For neurogenesis, KP2 and KP3 starting at 2 µg/mL and KP1 starting at 4 µg/mL significantly restored βIII-tubulin and MAP-2 mRNA expression in Aβ42-treated NE-4C cells in monoculture. In co-culture, all three KP fractions at 4 and 8 µg/mL induced βIII-tubulin and MAP-2 mRNA expression.
**Clinical Implications:** This study provides the first evidence that KP extracts, particularly the crude ethanolic (KP1), hexane (KP2), and chloroform (KP3) fractions containing methoxyflavones (DMF, PMF, TMF), can protect neural stem cells and neurons from Aβ42-induced damage through anti-inflammatory and antioxidant mechanisms. The extracts also counteracted Aβ42's inhibitory effects on neuronal differentiation. These findings suggest KP extracts may have therapeutic potential for AD by targeting both neuroinflammation and impaired neurogenesis. However, the study is limited to in vitro models, and further research is needed to identify which specific methoxyflavones are responsible for the observed effects and to elucidate the molecular pathways involved, particularly ERK1/2, PKA, Akt, and BDNF signaling. The NOAEL of >249 mg/kg BW/day from previous sub-chronic toxicity studies supports KP's safety profile for potential therapeutic development.