Quantitative Analysis and Biological Efficacies regarding the Neuroprotective and Antineuroinflammatory Actions of the Herbal Formula Jodeungsan in HT22 Hippocampal Cells and BV-2 Microglia

Jodeungsan (JDS) is a traditional herbal formula that comprises seven medicinal herbs and is broadly utilized to treat hypertension, dementia, and headache. However, the effects of JDS and its herbal components on neurodegenerative diseases have not been reported. We examined the inhibitory effects of JDS and its seven components on neuronal cell death and inflammation using HT22 hippocampal cells and BV-2 microglia, respectively. Among its seven herbal components, Uncaria sinensis (US), Chrysanthemum morifolium (CM), Zingiber officinale (ZO), Pinellia ternata (PT), Citrus unshiu (CU), and Poria cocos (PC) exhibited significant neuroprotective effects in HT22 cells. In BV-2 cells, JDS significantly suppressed the production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), indicating the antineuroinflammatory activity of JDS. In addition, the herbal extracts from ZO, Panax ginseng (PG), PT, CU, and PC exhibited inhibitory effects on the inflammatory response in microglia. These data imply that the JDS effect on neurodegeneration occurs via coordination among its seven components. To establish a quality control for JDS, a simultaneous analysis using five standard compounds identified hesperidin (37.892 ± 1.228 mg/g) as the most abundant phytochemical of JDS. Further investigation of the combinatorial activities of two or more standard compounds will be necessary to verify their antineurodegenerative regulatory mechanisms.


Introduction
Neurodegenerative disease is a medical condition that is characterized by the progressive loss of neural tissues. Parkinson's, Alzheimer's, and Huntington's diseases and their related disorders are neurodegenerative diseases [1,2]. Aging is one of the strongest risk factors for neurodegenerative diseases [3]. Senior populations are increasing in modern society, which leads to the increase in the prevalence of neurodegenerative diseases. Thus, these diseases are considered as significant challenges for both preclinical and clinical investigations. However, although abundant studies have tried to develop antineurodegeneration drugs, there are no powerful and effective agents; therefore, these diseases are currently incurable. Because the pathogenesis of neurodegenerative diseases is complex and neuronal and nonneuronal cells in neurovascular cell units coordinately participate in disease progression [4,5], a single molecule that targets these conditions is not a suitable approach in new drug development for neurodegenerative diseases. Recently, herbal medicines have been regarded as appropriate materials based on their positive aspect of having multiple components and multiple targets [6,7]. Several articles have suggested the possibility that herbal medicines are useful therapeutic agents for neurodegenerative diseases [8][9][10].
Jodeungsan (JDS), also called Chotosan in Japan and Diaoteng San in China, is a traditional herbal formula that Clinically, JDS has been prescribed for age-related diseases such as hypertension [11], dementia and cognitive impairment [12,13], and chronic headache [14]. Several clinical reports demonstrated the potential of JDS for treating dementia, one of the most common neurodegenerative diseases. Suzuki et al. reported that a double-blinded, randomized, and placebo-controlled study addressed the positive aspect of JDS on cognitive function and activities of daily living in patients with dementia [12]. Yamaguchi et al. reported that JDS improved the event-related brain potential in stroke patients with cognitive impairment [15]. Pharmacobiological evidence of the action of JDS against dementia has been described in in vitro or in vivo models [16][17][18]. However, most articles have described the anti-dementia effect of JDS itself, but not that of each of its herbal components.
Our study aimed to investigate the effect of JDS and its seven herbal components on neurodegenerative events using hippocampal and microglia cell lines. We also performed a simultaneous analysis of five standard compounds to improve the quality control of JDS.

Chemicals and Reagents.
The standard components, luteolin and apigenin, were purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, China), and hesperidin, 6-gingerol, and narirutin were purchased from Biopurify Phytochemicals (Chengdu, China). The chemical structures of the standard components are shown in Figure 1. The purity of these standard components was ≥98.0%, as assessed using high-performance liquid chromatography (HPLC) analysis. The HPLC-grade solvents, acetonitrile and water, were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ, USA), and the analytical-grade reagent trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Apparatus and Chromatographic
Conditions. The quantitative analysis was conducted using a Waters Alliance e2695 system (Waters Corp, Milford, MA, USA) equipped with a pump, degasser, column oven, autosampler, and photodiode array detector (#2998; Waters Corp). The data were acquired and processed using Empower software (version 3; Waters Corp). The chromatographic separation of the seven bioactive components was carried out at room temperature using Luna C 18 analytical columns (250 × 4.6 mm, 5 m), supplied by Phenomenex (Torrance, CA, USA), with a gradient solvent system of 0.1% (v/v) aqueous TFA (A) and acetonitrile (B). The elution conditions were as follows: 15%-40% B for 0-30 min, 40%-100% B for 30-40 min, and 100% B for 40-47 min. The flow rate was 1.0 mL/min and the injection volume was 10 L. The ultraviolet (UV) wavelength for

Calibration Curve and Determination of the Limits of Detection and Quantification.
The calibration curves of all components were calculated from the peak areas of the standard solutions at different concentrations. The tested concentration ranges were as follows: narirutin (12.5-400 g/mL), hesperidin (31.25-500 g/mL), luteolin (1.5625-25 g/mL), apigenin (0.78125-25 g/mL), and 6gingerol (3.125-50 g/mL). These solutions were measured in triplicate for the calibration curves. The limit of detection (LOD) and limit of quantification (LOQ) for the five standard components were calculated, using the slope of the calibration curve and the standard deviation (SD) of the intercept, as follows: LOD = 3.3 × (SD of the response/slope of the calibration curve); and LOQ = 10 × (SD of the response/slope of the calibration curve).

Herbal Extract Treatment and Cytotoxicity
Assay. BV-2 and HT22 cells were maintained in Dulbecco's modified Eagle's medium (Hyclone/Thermo, Rockford, IL, USA), supplemented with 10% fetal bovine serum (Hyclone/Thermo) and penicillin/streptomycin in a 5% CO 2 at 37 ∘ C. HT22 cells were cotreated with the herbal extract and hydrogen peroxide (H 2 O 2 , 250 M; Sigma-Aldrich) for 6 h. BV-2 cells were pretreated with various concentrations of each herbal extract for 2 h and treated with lipopolysaccharide (LPS, 1 g/mL; Sigma-Aldrich) for additional 22 h. The cytotoxicity test was performed as described previously [19]. In brief, BV-2 and HT22 cells were plated on 96well microplates at a density of 3 × 10 4 /well and 5 × 10 3 /well, respectively. Cells were treated with various concentrations of each herbal extract for 24 h. Cell counting kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added, and the cells were incubated for 4 h. The absorbance was read at 450 nm on an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The cell viability was calculated using the following equation: (1)

Measurement of Cytokine Production.
Culture supernatants were collected from the cells treated with various concentrations of each herbal extract in the presence of LPS. An enzyme-linked immunosorbent assay (ELISA) kit from R&D systems (Minneapolis, MN, USA) was used in accordance with the manufacturer's instructions. The concentration of each sample was calculated according to the standards provided with the kits.
2.9. Statistical Analysis. The data are expressed as the mean ± SEM. Data were analyzed using one-way analysis of variance and Dunnett's multiple comparisons test. < 0.05 was considered significant.

Optimization of HPLC
Separation. An HPLC analytical method was established for the simultaneous separation of the five standard components from the 70% ethanol extract of JDS. As a result, a completely separated chromatogram was obtained within 40 min using two mobile phases consisting of 0.1% (v/v) aqueous TFA (a) and acetonitrile (b). The UV wavelength used for quantitative analysis was 236 nm for narirutin, hesperidin, and 6-gingerol and 263 nm for luteolin and apigenin. Under these established HPLC methods, the retention times of narirutin, hesperidin, luteolin,

Compound
Linear range ( g/mL) Regression equation     Table 2. The calibration curves for the five components showed good linearity ( 2 ≥ 0.9998). The LOD and LOQ for the tested components were 0.013-1.054 g/mL and 0.041-3.194 g/mL, respectively.

Determination of the Five Standard Components of the JDS Extract.
The HPLC analytical method established here was applied to the simultaneous quantification of the five components of the extracted JDS sample. The relative amount of the five standard components ranged from 0.061 to 37.892 mg/g and the results of the analysis are summarized in Table 3. Among the five components of JDS, hesperidin was the most abundant.

Inhibitory Effect of JDS and Its Seven Herbal Components on Proinflammatory Cytokine Production in LPS-Stimulated
BV-2 Microglia. Next, we investigated whether JDS and its seven herbal components suppress the production of proinflammatory cytokines, such as tumor necrosis factoralpha (TNF-) and interleukin-6 (IL-6), in LPS-stimulated BV-2 cells. As shown in Figures 4 and 5, TNF-and IL-6 levels increased in the culture media of LPS-stimulated BV-2 cells, and these increases were significantly reduced by the treatment with JDS (Figures 4(a) and 5(a)) and its components ZO (d), PG (e), PT (f), CU (g), and PC (h). PC had the most significant inhibitory effect on the LPSstimulated TNF-and IL-6 production (Figures 4(h) and 5(h)).

Discussion
Neuronal cell death plays a crucial role in neurodegeneration [20]. In general, the induction of neuronal cell death in neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, is controlled by pathways related to mitochondrial dysfunction and oxidative stress [21,22].
Neuronal cell death is associated with neuroinflammation in the central nervous system [23]. Microglia are mainly involved in neuroinflammatory reaction mediated by environmental or microbial stimuli. Activated microglia promote the production of neurotoxin(s), thus leading to neuronal cell death [24]. In our study, we explored the effects of JDS and its seven herbal components on neurodegeneration. First, the neuroprotective effects of herbal extracts of JDS and its seven components were determined using the HT22 hippocampal cell line. To induce neuronal cell damage, HT22 cells were exposed to H 2 O 2 according to the accumulating evidence of H 2 O 2 -mediated neuronal cell death and alternation of redox signaling [25][26][27]. In the presence of concurrent treatment with H 2 O 2 and each of the herbal extracts, JDS had a significant effect of inhibition of neuronal cell death in H 2 O 2 -treated hippocampal cells. Its components US, CM, ZO, PT, CU, and PC also displayed neuroprotective effects that were concentration dependent. US treatment significantly reversed cell viability at lower concentrations (90.91% ± 3.42% and 82.78% ± 4.97% at 12.5 and 25 g/mL, resp.) compared with H 2 O 2 -treated cells (53.00% ± 3.92%). However, cotreatment with H 2 O 2 and a higher concentration of US significantly increased the level of H 2 O 2 -induced cell death. ZO significantly blocked neuronal cell death at 2.5 and 5 g/mL of the treatment (97.96% ± 13.57% and 84.74% ± 11.07%, resp.) compared with the H 2 O 2 control (55.20% ± 2.48%). The effect of PC was not strong compared to that of the other extracts. In cells that were cotreated with H 2 O 2 and PC at 25 g/mL, a 1.58-fold increase was observed compared to that observed for the treatment with H 2 O 2 alone (76.83%± 2.45% versus 48.53% ± 3.80%). CM, PT, and CU significantly protected against H 2 O 2 -induced cell death at a concentration ranging from 25 to 100 g/mL.  Microglia that are activated by neuronal injury stimulate the release of inflammatory cytokines, such as TNF-and IL-6 [24,28]. Thus, we assessed the production of TNFand IL-6 to examine the antineuroinflammatory activities of JDS and its herbal components. The inflammatory reaction was stimulated by adding LPS to microglia. As expected, LPS stimulation significantly enhanced the production of the cytokines TNF-and IL-6. The herbal mixture JDS significantly reduced the LPS-stimulated increase in cytokine generation observed in BV-2 cells. Among the seven components, ZO, PG, PT, CU, and PC had inhibitory effects on the production of TNF-and IL-6, and PC had the most dramatic effect of all the drugs tested.
Overall, our data demonstrate that JDS has neuroprotective and antineuroinflammatory effects. Among its seven components, ZO, PT, CU, and PC were commonly effective regarding both neuroprotection and anti-inflammation. CM and US were effective regarding neuroprotection, but not antineuroinflammation, whereas PG inhibited the inflammatory reaction with no neuroprotective activity. These data suggest that the combination of each component in the JDS herbal formula can accelerate the pharmacological activity of the drugs against neurodegeneration via a synergistic interaction.
In conclusion, the in vitro or in vivo efficacies of each standard compound of JDS on neurodegenerative diseases, including Alzheimer's disease, have been reported [38][39][40][41]. However, the combinatorial effect of the standard chemicals has never been considered. Further studies will be necessary to address the advantages of using herbal formulas compared to the administration of single herbs. In addition, the investigation of the efficacy of JDS should be expanded over various neurodegenerative diseases, as, to date, it has focused on dementia or the cognitive disorders of neurodegenerative diseases. The molecular mechanisms that underlie their effects should also be studied to confirm their potential as therapeutic agents for neurodegenerative diseases.