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. 2025 Aug 9;34(15):3631–3644. doi: 10.1007/s10068-025-01971-3

HPLC/UV analysis of mulberry vinegar and its protective role in LPS-induced cognitive impairment via regulating NF-κB signaling pathway and amyloidogenesis in mice

Se In Bang 1, Weon Taek Seo 2, Sanghyun Lee 3, Eun Ju Cho 1,✉, Ah Young Lee 4,✉
PMCID: PMC12528536  PMID: 41113276

Abstract

The present study investigated the protective effect of mulberry vinegar (MV) on inflammatory responses and cognitive deficit induced by lipopolysaccharide (LPS) in mice models. The mice were administered MV and given intraperitoneal injection of LPS. In behavioral tests, MV ameliorated memory deficit and cognitive dysfunction. In the LPS-injected mouse brains, the generation of malondialdehyde, reactive oxygen species, nitric oxide, and pro-inflammatory cytokines was inhibited by the administration of MV. These preventive roles of MV were attributed to inactivation of NF-κB signaling pathway with downregulation of iNOS and COX-2. Moreover, MV modulated the amyloidogenic pathway by inhibiting amyloid precursor protein, beta-site APP cleaving enzyme-1, presenilin 1, and presenilin 2 and enhancing Aβ degradation-related proteins expression. The major phenolic compounds in MV were protocatechuic acid (0.13 mg/mL), rutin (0.13 mg/mL), and chlorogenic acid (0.05 mg/mL), which were increased by fermentation, confirmed by HPLC/UV analysis. Therefore, MV could ameliorate cognitive deficits through regulation of oxidative stress, inflammatory responses, and amyloidogenesis.

Keywords: Alzheimer’s disease, Cognitive impairment, Mulberry, Neuroinflammation, Vinegar

Introduction

Alzheimer’s disease (AD), a predominant form of dementia, is accompanied by progressive memory and cognitive decline. The prevalence of AD is increased owing to extended life expectancy, and AD accounts for 70–80% of dementia cases in individuals over 65 years of age worldwide (Ramzan et al., 2020). The characteristic features of AD include accumulation of β-amyloid (Aβ) plaques, formation of neurofibrillary tangles, neuroinflammation, and the loss of neurons (Serrano-Pozo et al., 2011). Growing evidence indicates pathogenesis of AD extends beyond neuronal dysfunction and is closely linked to immune responses in the central nervous system (CNS) (Heneka et al., 2015). Neuroglia, the resident macrophages in the brain, are major immune effector cells that play crucial roles in the occurrence and development of neuroinflammation (McGeer et al., 1988). Although glial activation is responsible for host defense, excessive and prolonged stimulation of these cells can lead to neuronal death and the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, which upregulate β-secretase activity (Cameron and Landreth, 2010; Liu and Hong, 2003). In addition, chronically activated glial cells promote neuronal cell death by releasing highly toxic products such as malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide (NO) thereby enhancing amyloid precursor protein (APP) production and amyloidogenic processing (Krause and Muller, 2010; Li et al., 2013). Therefore, the reduction of these inflammatory factors through the inhibition of abnormal glial activation may be a putative therapeutic approach for neurodegenerative diseases, including AD.

Lipopolysaccharide (LPS), an immunostimulatory component of cell walls in gram-negative bacteria, facilitates inflammatory responses in the brain via the toll-like receptor-4 (TLR4)-mediated signaling pathway (Beutler, 2000). LPS was initially recognized as a TLR4 ligand, and TLR4 is mainly expressed on glial cells in the brain. Activated glial cells stimulated by LPS produce pro-inflammatory cytokines through nuclear factor-κB (NF-κB) signaling pathway (Zhang and Ghosh, 2000). NF-κB, belonging to the Rel family, plays a key role in transcription of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines. Additionally, LPS induces lipid peroxidation, ROS accumulation, and consequently causing oxidative injury in the brain (Barron et al., 2017; Pepe et al., 2018). A number of studies have demonstrated that the promoter region of beta-site APP-cleaving enzyme 1 (BACE1) gene contains NF-κB binding site, suggesting that NF-κB activation is closely associated with amyloidogenesis (Sambamurti et al., 2004; Sheng et al., 2003). Based on these findings, rodents intraperitoneally injected with LPS are commonly used as an experimental model for investigating interactions among inflammation, brain functions, and memory deficits in AD (Hennigan et al., 2007; Shaw et al., 2001). Several studies have shown a correlation between the use of anti-inflammatory agents and reduced incidences of AD. For instance, certain non-steroidal anti-inflammatory drugs (NSAIDs) have been evaluated as “γ-secretase modulators” related to Aβ deposition (Eriksen et al., 2003; Vlad et al., 2008). However, these synthetic medications have exhibited adverse gastrointestinal effects (Frolich, 2002); thus, any natural substance with anti-neurotoxicity and anti-inflammatory properties can be a good candidate for AD therapy.

Vinegar is one of the oldest products in the world composed of dilute acetic acid solution that is produced from any fermentable sugar substrate using various fermentation methods. Several studies have evaluated the functionality and pharmacological effects of vinegar, such as antimicrobial activity, prevention of inflammation and hypertension, and reduction of serum cholesterol (Budak et al., 2014; Murooka and Yamshita, 2008). These days, a wide range of vinegar products are produced using diverse raw materials and processing techniques to serve various purposes. Bouazza et al. (2016) observed that fruit vinegars exhibit antioxidant properties owing to their phenolic compounds and possess protective effect against liver damage in induced by high-fat diet in rats. Consequently, the interest of consumers in fermented vinegar derived from natural products has increased.

Mulberry (Morus alba L.), a representative red-colored berry fruit in Korea, contains abundant proteins, lipids, carbohydrates, fibers, minerals, and vitamins with low calories (Kim et al., 2010a). It has been consumed primarily as processed products such as jam, beverages, and liquor because of its low storability (Choi et al., 2006). Although there have recently been many efforts to use mulberry extract as a healthy functional food material instead of processed products (Guo et al., 2013; Jiao et al., 2017), studies on the neuroprotective and anti-inflammatory effects of mulberry vinegar (MV) remain limited. In our previous study, MV produced by alcoholic and acetic fermentation of mulberry juice (MJ) possessed in vitro radical scavenging effects (Bang et al., 2020). Furthermore, MV significantly attenuated LPS/IFN (interferon)-γ-induced inflammation in C6 glial cells by modulating glial activation and inhibiting NF-κB signaling pathway (Bang et al., 2022), suggesting that MV possesses both antioxidant and anti-inflammatory potential. Thus, we hypothesized that MV may exert neuroprotective effects in vivo by alleviating cognitive impairment and neuroinflammation. The present study was conducted to investigate whether MV could attenuate LPS-induced cognitive dysfunction in a mouse model, and to explore its underlying mechanisms related to inflammatory responses and amyloidogenesis.

Materials and methods

Sample preparation

Mulberry vinegar (MV) was produced using a two-step fermentation process as described in our previous study (Bang et al., 2020). Initially, mulberry fruits (Hadong, Republic of Korea) were crushed using a blender and 0.4% of Plantase™ (Bision Co., Seoul) was added. This mulberry juice (MJ) was then fortified with sugar to reach 15°Brix. For alcohol fermentation, Saccharomyces cerevisiae KCCM 11215 was introduced to the MJ and incubated at 30 °C for 8 days. After filtering using Whatman No. 1 filter paper, Acetobacter pasteurianus A8 was added to the MJ for subsequent acetic acid fermentation, which was then incubated with shaking at 30 °C for 20 days. The total acidity and pH of the MV were 4.26% and 3.50, respectively. The MJ and MV were stored at 4 °C for further experiments.

Animal experiment

Male ICR mice (5 weeks, 27–30 g) were obtained from Orient Inc. (Seongnam, Korea). Mice were kept in on a 12-h light–dark cycles with 20 ± 2 °C temperature and 50 ± 10% humidity. After a week of acclimation, the mice were divided into the following treatment groups: Normal = 0.9% NaCl (i.p); Control = LPS (0.25 mg/kg/day, i.p); MJ = LPS (0.25 mg/kg/day, i.p) and mulberry juice (1.5 mL/kg/day, oral gavage); MV = LPS (0.25 mg/kg/day, i.p) and mulberry vinegar (1.5 mL/kg/day, oral gavage); and DO = LPS (0.25 mg/kg/day, i.p) and donepezil (DO, 5 mg/kg/day, oral gavage). All mice were allowed unrestricted access to a standard diet (#5L79, Lab Diet, Orient Inc.) and MJ, MV, and DO were given at a daily volume of 100 μL. For the normal and control groups, an equivalent volume of drinking water was provided via oral gavage. In this study, the experimental dose of MJ or MV for mice was 1.5 mL/kg/day, which corresponds to a human equivalent dose of approximately 7.2 mL/day for adults with body weight of 60 kg. All experimental procedures were approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2020–2069), and the experimental schedule is illustrated in Fig. 1A.

Fig. 1.

Fig. 1

Fig. 1

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Effect of MJ and MV on cognitive dysfunction in LPS-injected mouse. Experimental schedule of behavioral tests (A). Initial latency and step-through latency in the passive avoidance test (B), spatial alternation in the T-maze test (C), object exploration rate (D), and discrimination index (E) in the novel object recognition test. Representative swimming-tracking pathways (F), percent of time to stay target quadrant (G), and escape latency to the hidden/exposed platform in the Morris water maze test (H). Values are mean ± SD (n = 7). a~cThe different letters among groups represent significant differences (P < 0.05) by Duncan’s multiple range test. *The cognitive abilities on space/object perceptive ability for old and new route (familiar and novel object) are significantly different as determined by Student’s t-test (P < 0.05). NS: Non-significance. Normal: 0.9% NaCl + intragastric administration of water; Control: 0.25 mg/kg/day LPS + intragastric administration of water; MJ: 0.25 mg/kg/day LPS + intragastric administration of mulberry juice (1.5 mL/kg/day); MV: 0.25 mg/kg/day LPS + intragastric administration of mulberry vinegar (1.5 mL/kg/day); DO: 0.25 mg/kg/day LPS + intragastric administration of donepezil (5 mg/kg/day)

Passive avoidance test

Passive avoidance is a fear-motivated test widely used as a simple memory-testing method by detecting through step-through apparatus composed of two chambers, which have an illuminated and dark chamber with a retractable door (Newman and Kosson, 1986). The floor in both compartments was made of metal rods 3 mm in diameter, spaced 1 cm apart (LE 100–26, Panlab, Spain). In the training trial, the mice were placed individually in the light compartment, and 10 s later, the door was opened. Mice tend to prefer darkness; thus, when the mice had completely moved into the dark compartment, the door was automatically closed and they were given electrical foot shock (0.5 mA, 3 s duration). After 24 h, the retention test was performed as on the previous day, and the time taken for the mice to enter the dark compartment, defined as “step-through latency (STL),” was measured with a 300 s cut-off time limit. If a mouse failed to cross the dark compartment within 300 s, it was considered to have retained memory of acquisition trial.

T-maze test

To assess spatial memory ability, the T-maze test was implemented according to the procedure established by Montgomery (1952). The T-maze apparatus consisted of a long start arm (length, 55 cm; width, 10 cm; height, 16.5 cm) and two short goal arms (each length, 30 cm; width, 10 cm; height, 16.5 cm) in opposite directions. In the training session, each mouse was permitted to enter only one arm (familiar route) for 10 min in the T-maze. Twenty-four hours later (test session), the mice were encouraged to explore both familiar and unfamiliar arms for 10 min. Entries into each arm were recorded, and spatial recognition ability (%) was determined by calculating the percentage of entries into the new arm relative to the total maze entries. It was judged that the higher the detection rate for the new pathway, the better the memory of the mouse.

Novel object recognition test

The novel object recognition test was conducted to evaluate the recognition memory (Bevins and Besheer, 2006). During the training session, two identical objects were positioned within a square open-field shaped black box (45 × 45 × 45 cm), and each mouse was habituated at the center of the field. Mice were allowed to interact with two objects for 10 min, and the frequency of times they touched each object was measured during this period. After 24 h, one of the two objects was replaced with a different shape and size object and the mice were given 10 min to explore both objects. The number of touches on each object was counted during both training and test trials. Object exploration rate (%) was calculated as the number of touches on each object relative to the total number of touches on both objects, which was multiplied by 100. Discrimination index (DI) of the test session reflects the degree of preference for the novel object and is calculated as follows: [(number of touches on novel object – number of touches on familiar object) / total number of touches on both objects] × 100 (Choi et al., 2023; Wang et al., 2021).

Morris water maze test

The Morris water maze test is a commonly accepted method for examining cognitive function based on the experimental scheme of Morris (1984), with some slight modifications. In Morris water maze test, each mouse was monitored using the SMART-LD program (Panlab, Barcelona, Spain). The test was performed in a 150 cm-diameter stainless steel circular pool filled with squid ink water (22 ± 2 °C), and the pool was divided into four-equal sized quadrants with different visual cues as a navigator. A hidden escape platform was located at the midpoint of one quadrant. During the acquisition phase (days 1–3), mice underwent three trials/day of hidden platform test to learn how to use visual cues surrounding the pool to locate the submerged platform. Each trial lasted up to 60 s or ended when the mouse successfully reached the hidden platform. If they did not find the target platform, they remained there for 15 s to familiarize themselves with platform’s location and associated visual cues. On the final day of training (day 4), probe tests were conducted. The first test followed same protocol as the training trials. In the second test, the target platform was removed from the water, and duration of time spent on target quadrant, where the platform had been previously located, was used as a measure of memory retention. For the final test, the target platform was placed in a pool filled with transparent water, and the time taken by the mice to reach the visible platform was recorded.

Measurement of lipid peroxidation

The production of MDA, which is the main breakdown product of oxidized lipids, was analyzed following the protocol developed by Ohkawa et al. (1979). The collected brain tissues were homogenized in 0.9% NaCl solution (LPS solution, Seoul, Korea) and added to 46 mM TBA (Ward Hill, MA, USA) and 920 mM TCA solutions (Sigma Aldrich Inc.). The mixture was boiled for 20 min. The MDA produced was determined at 540 nm using a microplate reader (Rayto Life and Analytical Sciences Co. Ltd.).

Measurement of ROS generation

The ROS level in the brain tissue was assessed following a method of Muhammad et al (2019). In brief, homogenized brain tissues were reacted with 12.5 mM 2’,7’-dichlorofluorescein diacetate (DCF-DA) solution during 10 min, and the levels of ROS were quantified by measuring fluorescence (Ex: 485 nm, Em: 535 nm) using a FLUOstar OPTIMA fluorescence (BMG Labtech, Ortenberg, Germany).

Measurement of NO formation

The NO levels in the brain tissue were detected using colorimetric assay based on the reaction of NO with Griess reagent (Tsikas, 2007). After homogenization of brain tissue, an equivalent volume of Griess reagent was added to reaction, and then incubated at room temperature for 30 min in the dark. Absorbance was measured at 540 nm using a microplate reader (Rayto Life and Analytical Sciences Co. Ltd.), and NO concentration was calculated using a standard curve prepared with NaNO2 (ranging from 1 to 100 μM).

Quantification of inflammatory cytokines by enzyme-linked immunosorbent (ELISA) assay

The frozen brains were homogenized in 100 mg tissue/mL lysis buffer. The homogenates were centrifuged at 3000 rpm for 15 min, and the supernatant was collected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β in the supernatant were determined using ELISA kits (R&D Systems, USA) in accordance with the manufacturer’s instructions.

Western blotting

The brain tissues were lysed using RIPA buffer supplemented with 1X protease inhibitor cocktail. After quantifying the proteins via BIO-RAD assay, equal amounts of protein were resolved on 8–13% sodium dodecyl sulfate–polyacrylamide gel followed by transferring the proteins from the gels to PVDF membranes and blocking with 5% skim milk. To detect target proteins, the membranes were incubated with specific primary antibodies and appropriate secondary antibodies. The protein bands were scanned using a chemiluminescent imaging system (Davinch Chemi, Seoul, Korea).

HPLC analysis and calibration curves

Protochatechuic acid (1), chlorogenic acid (2), cyanidin-3-O-glucoside (3), cyanidin-3-O-rutinoisde (4), rutin (5), quercetin (6) were obtained from the Natural Product Institute of Science and Technology (www.nist.re.kr) in Anseong, Republic of Korea. For quantitative analysis, 50 mg of freeze-dried MJ and MV powder, along with 1 mg of compounds 1–6, were accurately weighed, dissolved in 1 mL of methanol, sonicated for 20 min, and filtered through a 0.45 μm PVDF membrane filter (Cat No. 6779, NJ, USA). The analysis was performed using a reverse-phase HPLC system (Agilent 1260 Infinity II Quat Pump, CA, USA) equipped with a YMC Pack-Pro-C18 column (25 cm × 4.6 mm, 5 μm). The mobile phase consisted of 0.1% formic acid in water (A) and a mixture of water, acetonitrile, and formic acid in a ratio of 60:30:10 (B), with gradient elution conditions set as follows: 20% B for the initial 10 min, 40% B at 10 min, 50% B at 25 min, 100% B from 26 to 30 min, and returning to 20% B from 35 to 45 min. The column temperature was maintained at room temperature, the injection volume was set at 10 μL, the flow rate was 1 mL/min, and detection was conducted at a wavelength of 280 nm. Standard stock solutions of compounds 1–6 were prepared and serially diluted to concentrations ranging from 0.977 to 250 μg/mL to construct calibration curves, which were established by plotting the peak area (Y) against the concentration (X, μg/mL) using the mean values (n = 3) ± standard deviation (SD). Linearity was assessed based on the correlation coefficient (r2), and the contents of compounds 1–6 in the samples were quantified accordingly.

Statistical analysis

In this study, the results were presented as the means ± SD. Statistical differences between groups were determined by one-way ANOVA and Duncan’s multiple range test using the SPSS statistical program (version 25, SPSS Inc., NY, USA). In behavioral tests (passive avoidance test, T-maze test, and novel object recognition test), Student t-test was used to analyze the comparing the training and test session. Statistical significance was considered at P < 0.05.

Results and discussion

AD, one of the most prevalent neurodegenerative disorders, is characterized by the the accumulation of Aβ plaques, which leads to memory loss and cognitive decline (Ittner and Götz, 2011). Neuroinflammation in the CNS plays a critical role in the pathophysiology of AD, with several neuroinflammatory factors contributing to its onset and progression (Rubio-Perez and Morillas-Ruiz, 2012). Furthermore, epidemiological research has shown that the use of anti-inflammatory drugs is associated with a reduced risk of AD (Szekely et al., 2004). Treatment with LPS, an endotoxins derived from gram-negative bacteria, induces neuroinflammation along with blood–brain barrier (BBB) damage, thereby causing amyloidogenesis and memory deficiency (Herber et al., 2004). According to these reports, the LPS-induced mouse model is widely used for identifying the mechanisms of neurodegeneration and testing potential therapeutic substances (Noh et al., 2014; Zarifkar et al., 2010). Therefore, an in vivo LPS-induced model was employed to investigate whether MV supplementation could suppress neuroinflammation, amyloidogenesis, and cognitive impairment.

Mulberry has been generally consumed as a folk medicine in China and other East Asian countries for sore throat treatment, fever removal, or improvement of eye functions (Yuan and Zhao, 2017; Zhou et al., 2017). Mulberry contains abundant bioactive compounds such as anthocyanins, polyphenols, and flavonoids, which exhibit various biological activities including antioxidant, anti-atherosclerosis, immunomodulatory, neuroprotective, and other activities (Chang et al., 2013). Kang et al. (2006) described the neuroprotective effect of cyanidin 3-O-beta-D-glucopyranoside from mulberry fruits using a mouse model of brain injury with transient middle cerebral artery occlusion. Ethanol extract of mulberry fruit attenuated PD-like symptoms by inhibiting ROS and NO production, and the activation of caspase-3-dependent apoptosis pathway (Kim et al., 2010b). In addition, mulberry fruit extracts moderate brain damage and memory impairment in animal models (Kawvised et al., 2017; Liu and Du, 2020). However, mulberry is perishable because of its low production efficiency and high-water content and is therefore consumed only as fruit juice or jam. For this reason, this study aimed to produce MV through the fermentation of MJ and to evaluate its potential protective effects against cognitive impairment in LPS-injected mice.

The passive avoidance test has been widely used for testing memory impairment, which is based on the tendency of mice to prefer darkness (Nomura et al., 1994). Previous report confirmed that animals treated with LPS exhibit passive avoidance ability, remaining in the illuminated compartment for a shorter amount of time (Zhao et al., 2019). During the first day of training, there was no difference in the latency of entering the dark compartment. Following the given electric shock, the memory was observed the next day. Figure 1B illustrated that a shorter retention latency and reduced STL (37.29 ± 31.83 s) to enter the dark compartment entries were observed in the LPS-treated control group than in untreated group (288.30 ± 19.53 s), demonstrating that the LPS injection weakened memory consolidation in the mice. In contrast to the control group, the group administered MJ and MV exhibited improved STL, which was similar to that in the normal group, implying decrease the memory errors by treatment with MJ and MV.

The T-maze and novel recognition tests were performed to examine the short-term memory function of LPS-induced mice, depending on the innate preference for novelty in rodents (Deacon and Rawlins, 2006). In T-maze test, LPS-injected control group did not show any significant changes in the number of entries between new and old routes (Fig. 1C). In contrast, MJ-and MV-treated groups remarkably preferred to explore the new route rather than the old route (from 41.54 to 58.46% in MJ group, from 20.91 to 59.09% in MV group, respectively). We also observed that no significant difference between the number of times the mice touched the familiar object (49.88%) and novel object (47.08%) in mice injected with LPS, indicating that impaired ability to distinguish the new object from the familiar object (Fig. 1D). However, the MJ- and MV-administered mice were able to distinguish the novel object. Additionally, mice pre-treated with DO and subsequently injected with LPS were able to distinguish the novel object; they spent more time exploring the novel object (58.00%) than the familiar object (44.16%). The discrimination index was also decreased in the LPS-injected control group compared to the normal group, whereas it was significantly increased in the MJ- and MV-supplemented groups (Fig. 1E). These results suggest that MJ and MV effectively reversed LPS-induced novelty-based memory impairment.

The Morris water maze test is considered the most basic long-term spatial learning memory task, utilizing visual tips to discover the hidden target platform. LPS deteriorated long-term spatial learning performance in this test, exhibiting the longest escape latency to reach the target platform (Ham et al., 2019). Across multiple training sessions, the mice progressively learned to locate the hidden platform (Fig. 1F). LPS-induced mice took longer to find the platform compared to the normal group, indicating LPS-induced cognitive impairment. In contrast, mice administered MJ and MV demonstrated a reduction in the time required to reach the hidden platform, indicating that these groups found located the target platform more quickly than the LPS-induced control group. On the final day, a probe test was conducted to assess memory retention by measuring the time spent in the target quadrant (Fig. 1G). The LPS-induced control group exhibited a reduced occupancy in the target quadrant (13.35%) compared to the normal group (25%). However, the time spent in the target quadrant was significantly increased by treatment with treatment with MJ and MV, showing 20.66% and 19.50%, respectively, suggesting their beneficial effects in counteracting LPS-induced memory deficits. The latency to reach the visible platform was measured to assess whether mice performance is related to visual or motor functions. No significant differences were observed among the groups, confirming that these effects were not attributed by visual or motor abilities (Fig. 1H). Although no significant differences were noted between the effects of MJ and MV, both effectively mitigated LPS-induced cognitive impairments and exhibited similar efficacy to DO.

Neuroinflammation induced by LPS has been shown to trigger activation of glial cells, leading to the release of neurotoxic substances, including inflammatory cytokines and free radicals (Noworyta-Sokołowska et al., 2013). LPS caused the recruitment of neutrophils and macrophages, which in turn facilitates the production of inflammatory mediators such as NO and ROS (Li et al., 2013). To evaluate the protective effect of MV on oxidative stress and inflammatory responses in the brain of LPS-injected mice, the levels of MDA, ROS, NO, and pro-inflammatory cytokines were assessed. In mice brain, MDA level was significantly increased to 79.7 nmol/mg protein in LPS control group, whereas that of the normal group was only 63.8 nmol/mg protein (Fig. 2A). However, the MDA concentration was significantly decreased to 63.6 nmol/mg protein by pretreatment of MV, respectively. Moreover, in contrast to LPS-injected control group, MJ and MV group significantly inhibited ROS levels in mice brain (Fig. 2B). The concentration of NO in the brain of LPS-injected control mice (33.3 μmol/mg protein) was remarkably higher than that in the normal group (8.7 μmol/mg protein) (Fig. 2C). In contrast, the MJ- and MV-treated groups showed significantly lower NO level (15.3 μmol/mg protein and 15.9 μmol/mg protein, respectively) compared with the control group. In addition, oral gavage of DO induced a reduction in NO level to 19.3 μmol/mg protein. These results indicated that the LPS injection resulted in overproduction of NO, which was inhibited by pretreatment with MJ, MV, and DO. The production of TNF-α (Fig. 2D), IL-6 (Fig. 2E), and IL-1β (Fig. 2F) in the brain homogenates of the LPS-induced mice were higher than that in the normal group. In response to MJ and MV administration, diminished levels of TNF-α, IL-6, and IL-1β were observed. Particularly, the MJ and MV groups exhibited markedly restrained TNF-α production compared to DO groups. Therefore, these results indicated that oral administration of MJ and MV suppressed the release of pro-inflammatory cytokines and the occurrence of inflammation in LPS-induced mouse brains.

Fig. 2.

Fig. 2

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Effects of MJ and MV on MDA (A), ROS (B), NO (C), TNF-α (D), IL-6 (E), IL-1β (F) production levels in the brain of LPS-injected mouse. Values are mean ± SD (n = 7). a~dThe different letters among groups represent significant differences (P < 0.05) by Duncan’s multiple range test. Normal: 0.9% NaCl + intragastric administration of water; Control: 0.25 mg/kg/day LPS + intragastric administration of water; MJ: 0.25 mg/kg/day LPS + intragastric administration of mulberry juice (1.5 mL/kg/day); MV: 0.25 mg/kg/day LPS + intragastric administration of mulberry vinegar (1.5 mL/kg/day); DO: 0.25 mg/kg/day LPS + intragastric administration of donepezil (5 mg/kg/day)

Further experiments were conducted to understand the mechanisms through which MV regulates inflammatory responses in LPS-induced mice. NF-κB, a ubiquitous transcription factor, regulates several other genes involved in inflammatory responses (Lawrence, 2009). Stimulation with LPS induces neuroinflammation through the activation of NF-κB and leads to the overproduction of inflammatory cytokines, which has been observed in neurons and astrocytes in patients with AD (Kaltschmidt et al., 1997). TLR4 is an indispensable immune receptor for LPS, mediating several inflammatory pathways through a MyD88-dependent pathway. When LPS binds to TLR4, IκB kinase (IKK) is activated, causing the phosphorylation of IκB, followed by the translocation of cytoplasmic NF-κB p50/p65 from the NF-κB/IκB complex into the nucleus, consequently inducing several inflammatory genes such as TNF-α, IL-6, IL-1β, iNOS, and COX-2 (Kawai and Akira, 2007). Overexpression of COX-2 can impair memory ability and synaptic plasticity and upregulation of iNOS interrupts memory consolidation by altering cholinergic function (Kotilinek et al., 2008; Udayabanu et al., 2008). In addition, oxidative stress is responsible for activation of the NF-κB signaling pathway, resulting in NO and ROS production (Tak and Firestein, 2001). Therefore, modulating NF-κB-mediated cellular pathways could serve a promising therapeutic target for neurological disorders associated with neuroinflammation. Our results showed that the activities of TLR4, MyD88, NF-κB p65, and p-IκB-α were significantly increased in the LPS-treated control mice brain compared with those in the normal group (Fig. 3). However, the expression levels of these proteins were downregulated in the MJ and MV groups. The iNOS and COX-2 expressions were also upregulated in the LPS-injected group compared to those in the normal group without LPS injection. In contrast, the protein expressions of iNOS and COX-2 were significantly downregulated in the MJ- and MV-administered mouse brains, demonstrating that LPS stimulates the inflammatory reaction through activation of the NF-κB signaling pathway, which was noticeably alleviated by MJ and MV treatment. Although additional investigations are needed to confirm the specific mechanism underlying the anti-neuroinflammatory effects of MJ and MV, it is believed that the protective effects of MJ and MV on cognitive impairment were partly due to the inhibition of nuclear translocation of NF-κB in LPS-induced mice.

Fig. 3.

Fig. 3

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Effects of MJ and MV on protein expression levels of inflammation mediator factors in brain of LPS-injected mouse. Western blotting analysis of TLR4, MyD88, NF-κB p65, and p-IκB-α protein expression (A) and iNOS and COX-2 protein expression (B). Values are mean ± SD (n = 3). a~eThe different letters among groups represent significant differences (P < 0.05) by Duncan’s multiple range test. β-actin served as a loading control. Normal: 0.9% NaCl + intragastric administration of water; Control: 0.25 mg/kg/day LPS + intragastric administration of water; MJ: 0.25 mg/kg/day LPS + intragastric administration of mulberry juice (1.5 mL/kg/day); MV: 0.25 mg/kg/day LPS + intragastric administration of mulberry vinegar (1.5 mL/kg/day); DO: 0.25 mg/kg/day LPS + intragastric administration of donepezil (5 mg/kg/day)

During inflammation, Aβ deposition stimulates glial cells to produce inflammatory cytokines and chemokines, which inadvertently attack host neurons. Aβ is generated by sequential proteolysis of APP, a large integral membrane protein that is first cleaved by BACE, followed by γ-secretase, presenilin 1 (PS1) and presenilin 2 (PS2), in the transmembrane domain (Vassar et al., 1999). In the brains of patients with AD, activated NF-κB is predominant in glial cells surrounded by high amounts of Aβ plaques, implying that Aβ accumulation is closely related to neuroinflammatory responses (Terai et al., 1996). It has been reported that BACE promoter contains an NF-κB binding site, and thus increased expression of NF-κB p65 can activate BACE transcription, leading to elevated C99 levels and promoting amyloidogenesis (Sambamurti et al., 2004; Sheng et al., 2003). Numerous evidence indicate that LPS injection impairs cognitive function and enhances amyloidogenesis by activating the NF-κB signaling pathway (Gu et al., 2018; Liu et al., 2017). The decomposition and clearance of amyloid plaques is also gradually gaining attention as an alternative APP therapeutic approach. In particular, endogenous proteases neprilysin (NEP) and insulin-degrading enzyme (IDE) have been identified as key Aβ-degrading enzymes (Sikanyika et al., 2019). Pro-inflammatory cytokines, such as TNF-α, not only promote Aβ accumulation through increase of BACE expression but also suppress Aβ degrading enzymes, such as NEP and IDE (Yamamoto et al., 2007). Consistent with the above studies, the LPS-treated group exhibited considerably elevated expression levels of amyloidogenic proteins (APP, BACE, PS1, and PS2) compared to the non-treated normal group, thus elucidating that LPS induced Aβ generation (Fig. 4A). In contrast, MJ and MV administration significantly reduced these protein expressions, suggesting that MJ and MV may improve cognitive function by suppressing Aβ generation in the LPS-induced mouse brain by inhibiting the activity of β- and γ-secretase. Figure 4B illustrates the effects of MJ and MV on the expression of proteins related to the clearance and degradation of Aβ. The expression levels of IDE and NEP, which are responsible for the degradation of Aβ, were downregulated in the LPS-injected control brain, compared to the saline-treated normal brain. However, supplementation with MJ and MV resulted in significant upregulation of NEP and IDE expression, suggesting that MJ and MV may promote Aβ degradation in the LPS-injected mouse brain via regulation of IDE and NEP enzyme activity. These data demonstrated that the protective effects of MJ and MV against LPS-induced cognitive impairment were attributed to the inhibition of the amyloidogenic process and enhancement of Aβ degradation.

Fig. 4.

Fig. 4

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Effects of MJ and MV on protein expression levels of amyloidogenesis and Aβ-degrading enzyme in brain of LPS-injected mouse. Western blotting analysis of APP, BACE, PS1, and PS2 protein expression A and IDE and NEP protein expression B. Values are mean ± SD (n = 3). a~eThe different letters among groups represent significant differences (P < 0.05) by Duncan’s multiple range test. β-actin served as a loading control. Normal: 0.9% NaCl + intragastric administration of water; Control: 0.25 mg/kg/day LPS + intragastric administration of water; MJ: 0.25 mg/kg/day LPS + intragastric administration of mulberry juice (1.5 mL/kg/day); MV: 0.25 mg/kg/day LPS + intragastric administration of mulberry vinegar (1.5 mL/kg/day); DO: 0.25 mg/kg/day LPS + intragastric administration of donepezil (5 mg/kg/day)

The beneficial effects of MV may be associated with their bioactive compounds, including amino acids, organic acids, and phenolic substances, which are derived from its raw materials and may be generated or enhanced during the fermentation process. Additionally, phenolic compounds in MV can also undergo transformation into new bioactive molecules. As summarized in Table 1 and Fig. 5, six compounds were identified using an HPLC/UV system: protocatechuic acid (1), chlorogenic acid (2), cyanidin-3-O-glucoside (3), cyanidin-3-O-rutinoside (4), rutin (5), and quercetin (6). Interestingly, MV was found to be rich in protocatechuic acid (1), chlorogenic acid (2), and rutin (5), which phenolic compounds strongly associated with antioxidant and anti-inflammatory properties, compared to MJ. In MV, the concentrations of protocatechuic acid (1), rutin (5), and chlorogenic acid (2) were 0.13 mg/mL, 0.13 mg/mL, and 0.05 mg/mL, respectively, which were higher than those in MJ (0.10 mg/mL, 0.07 mg/mL, and 0.01 mg/mL, respectively). On the other hand, the levels of cyanidin-3-O-glucoside (3), cyanidin-3-O-rutinoside (4), and quercetin (6) were higher in MJ compared to MV, indicating that these compounds may have been metabolized during the fermentation process to facilitate bacterial growth and reproduction. Anthocyanins, which are natural pigments present in mulberry in glycoside form, were observed to decrease significantly in content, particularly cyanidin-3-O-glucoside (3) and cyanidin-3-O-rutinoside (4), when comparing MV to MJ. Cyanidin-based anthocyanins, such as cyanidin-3-O-glucoside (3) and cyanidin-3-O-rutinoside (4), are known for their structural sensitivity to acidic environments, temperatures, and enzymatic activity. During acetic acid fermentation, secreted β-glucosidase and other glycosidases from acetic acid bacteria can catalyze the hydrolysis of glycosidic bonds in anthocyanins. In contrast, the increase in rutin content is likely due to microbial biotransformation of quercetin precursors. In the present study, the content of quercetin (6) was found to decrease after fermentation, supporting that quercetin (6) or its derivatives may have been converted into rutin (5) via microbial enzymatic activity. Genomic analyses of certain A. pasteurianus strains have identified genes encoding glycosyltransferase-related domains, suggesting that these species may possess glycosylation capabilities during fermentation possess glycosyltransferase activity (Yamaura et al., 2024). Although no reports have demonstrated glycosyltransferase activity of A. pasteurianus A8 used in this study, further investigation on genomic sequencing and enzymatic assays would be required to verify enzymatic potential for glycosylation. Additionally, increasing the relative concentration of rutin (5) may be partly due to the degradation or loss of sugar. According to our previous study, sugars such as glucose and fructose are significantly consumed during the fermentation process, potentially contributing to shifts in the relative abundance of certain phenolic compounds, including rutin (5) (Bang et al., 2020). Protocatechuic acid (1) and chlorogenic acid (2) have demonstrated the ability to inhibit microglia activation and neuroinflammation-induced cognitive dysfunction in both in vitro and in vivo studies (Kaewmool et al., 2020; Xiong et al., 2023). Moreover, rutin (5) supplementation has been shown to prevent memory impairment by suppressing oxidative stress, Aβ and tau pathology, and inflammatory responses in an AD mouse model (Sun et al., 2021). Therefore, our results suggest that the phenolic compounds in MV, such as protocatechuic acid (1), chlorogenic acid (2), and rutin (5), may play a key role in its anti-inflammatory effects and its potential to improve cognitive abilities.

Table 1.

Quantitative analysis of compounds 1–6 in MJ and MV

Compounds Contents (mg/mL)
MJ MV
Protocatechuic acid (1) 0.10 ± 0.00 0.13 ± 0.00
Chlorogenic acid (2) 0.01 ± 0.00 0.05 ± 0.00
Cyanidin-3-O-glucoside (3) 0.02 ± 0.00 0.01 ± 0.00
Cyanidin-3-O-rutinoside (4) 0.04 ± 0.00 0.00 ± 0.00
Rutin (5) 0.07 ± 0.00 0.13 ± 0.00
Quercetin (6) 0.18 ± 0.00 0.07 ± 0.00
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Values are mean ± SD (n = 3)

Fig. 5.

Fig. 5

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HPLC/UV chromatograms of MJ (A) and MV (B). Chemical structures in MJ and MV (C); protochatechuic acid (1), chlorogenic acid (2), cyanidin-3-O-glucoside (3), cyanidin-3-O-rutinoisde (4), rutin (5), and quercetin (6)

In conclusion, MV supplementation could alleviate LPS-induced cognitive impairment in a mouse model, which might be related to the suppression of oxidative stress and regulation of inflammatory responses. Moreover, MV inhibited amyloidogenesis and enhanced Aβ degradation. These findings suggest that MV may be useful as a functional vinegar for consideration in the treatment of neurodegenerative diseases, such as AD.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2019R1G1A1100315)

Author contributions

S.I.B; Writing – original draft, Methodology, Investigation, Formal analysis. W.T.S; Investigation, Resources, Validation. S.L.; Formal analysis, Investigation, Methodology. E.J.C; Conceptualization, Supervision, Writing – review & editing. A.Y.L; Conceptualization, Data curation, Funding acquisition, Writing – original draft, Writing – review & editing, Project administration. All the authors have read and agreed to the published version of this manuscript.

Declarations

Conflict of interest

The authors state no conflict of interest.

Ethical approval

The animal study protocol was approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2020–2069) on the ethical procedures and scientific care of laboratory animals.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Eun Ju Cho, Email: ejcho@pusan.ac.kr.

Ah Young Lee, Email: aylee@gnu.ac.kr.

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