Protective Roles of Monsonia angustifolia and Its Active Compounds

Apr 5, 2017 - Council for Scientific and Industrial Research, Pretoria, South Africa. # Department of Chemistry, University of Pretoria, Private Bag X...
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Protective Roles of Monsonia angustifolia and Its Active Compounds in Experimental Models of Alzheimer’s Disease Yoon Sun Chun,†,§,∥ Joonki Kim,†,∥ Sungkwon Chung,§ Eric Khorombi,⊥ Dashnie Naidoo,⊥ Rudzani Nthambeleni,⊥ Nial Harding,⊥ Vinesh Maharaj,# Gerda Fouche,*,⊥ and Hyun Ok Yang*,†,Δ †

Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do, Republic of Korea Department of Physiology, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do, Republic of Korea ⊥ Council for Scientific and Industrial Research, Pretoria, South Africa # Department of Chemistry, University of Pretoria, Private Bag X20, Hatfield 0028, Pretoria, South Africa Δ Department of Biological Chemistry, University of Science & Technology (UST), Daejeon, Republic of Korea §

S Supporting Information *

ABSTRACT: Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is characterized by the accumulation of neurotoxic β-amyloid (Aβ) peptides, which consequently affects cognitive decline and memory impairment. Current research on AD treatment is actively focusing on the prevention of neurotoxic Aβ peptide accumulation. Monsonia angustifolia is reported to be consumed as an indigenous vegetable in Tanzania. In this study, we investigated the effect of the ethanol (EtOH) extract of M. angustifolia dried ground material on Aβ production and spatial learning ability as protection against AD. The formation of Aβ peptides was significantly reduced in HeLa cells stably transfected with the Swedish mutant form of β-amyloid precursor protein (APPsw) after treatment with a 60% EtOH extract of M. angustifolia. We next examined the cognitive-improving effects of the EtOH extract in vivo. Tg2576 mice were treated with extract for 6 months and subjected to Morris water maze and novel object recognition tests. The results showed that the 60% EtOH extract of M. angustifolia significantly ameliorated behavioral deficits of the AD transgenic mice and reduced the level of insoluble Aβ42 in the cerebral cortex and hippocampus. We further found that the 60% EtOH extract was effective for memory function recovery after shorter treatment (4 months). In addition, we isolated and identified several single compounds, justicidin A, 5-methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone, from M. angustifolia and tested these compounds. Among them, justicidin A potently decreased the formation of Aβ in APPsw-transfected cells. These data suggest that the 60% EtOH extract of M. angustifolia has the potential to be developed as a treatment of AD. Furthermore, justicidin A may contribute, at least partially, to the Aβ alteration observed with the extract treatment. KEYWORDS: Monsonia angustifolia, Alzheimer’s disease, β-amyloid, justicidin A, cognition-improving effect

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challenge to cross the blood−brain barrier.9 In addition, γsecretase inhibitors may cause side effects such as the failure of Notch signaling activation, which is essential for development and differentiation.10 In fact, γ-secretase modulators reduce the Aβ42 level by producing shorter Aβ species. Even though shorter Aβ species may be less toxic than longer Aβ species, they still have a possibility to induce neuronal dysfunction.11 Studies have shown that α-secretase is activated by protein kinase C activators and cholesterol-lowering drugs, statins.12,13 However, A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, known as α-secretases of APP, contribute to the proteolysis of APP as well as tumor necrosis factor-α and epidermal growth factor receptor ligands involved in inflammation and cancer.14 Thus, the modulation of secretases can limit the efficacy and safety as a treatment for AD-related Aβ pathology. Furthermore, FDA-approved AD drugs, such as acetylcholinesterase inhibitors and the N-methyl-D-aspartate

INTRODUCTION Alzheimer’s disease (AD), a neurodegenerative disorder, is pathologically characterized by the formation of cerebral plaques and neurofibrillary tangles in the brain. β-Amyloid (Aβ) is the major component of these senile plaques resulting in neurite dystrophy.1,2 Aβ is generated by the proteolysis of βamyloid precursor protein (APP), a type I transmembrane protein, through sequential cleavage by β- and γ-secretase.3 This is called the amyloidogenic pathway. The two major isoforms of Aβ, Aβ40 and Aβ42, were found in AD patients. Aβ42 is more prone to rapid aggregation in AD brains, whereas Aβ40 is an abundant isoform in the normal brains. 4 Alternatively, APP can be cleaved within the Aβ region by αsecretase, precluding the formation of Aβ, thus initiating the non-amyloidogenic pathway.5 Research on AD treatments has been developed to decrease Aβ levels by inhibiting or modulating β- and γ-secretase or to stimulate α-secretase toward the non-amyloidogenic pathway in APP processing.1,6 Thus, several β- or γ-secretase inhibitors and modulators have been designed.7,8 However, the developed βsecretase inhibitors, including peptidomimetic inhibitors and a variety of nonpeptide heterocyclic inhibitors, still confront a © XXXX American Chemical Society

Received: October 11, 2016 Revised: March 6, 2017 Accepted: March 21, 2017

A

DOI: 10.1021/acs.jafc.6b04451 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Relative Quantities of the Five Compounds Isolated from the 60% EtOH Extract and the MeOH/DCM (1:1) Extract of BP21a peak area (60% EtOH)

compound justicidin A 5-methoxyjusticidin A chinensinaphthol retrochinensinaphthol methyl ether suchilactone

peak area (MeOH/DCM)

ratio by peak area

retention time (min)

UV

ESI+

UV

ESI+

UV

ESI+

33.98 39.27 24.19 36.60

133567 177101 19651 26861

7153234 14154399 1021425 2012201

1503017 995692 981644 204366

33080586 31281136 16920828 16233100

0.08887 0.17787 0.02002 0.13144

0.21624 0.45249 0.06036 0.12396

29.87

27968

2679751

101073

7417652

0.27672

0.36127

av

quantity isolated from MeOH/DCM extract (mg/6 g extract)

estimated quantity from 60% EtOH extract (mg/6 g extract)

0.153 0.315 0.040 0.128

19 35 27 16

2.90 11.03 1.09 2.04

0.319

36

11.48

A comparison was made between the chemical profiles of the 60% EtOH and MeOH/DCM (1:1) extracts and the relative quantities of the five compounds determined in the 60% EtOH extract comparedwith those of the MeOH/DCM (1:1) extract. Relative quantification was performed by comparing the integrated peak areas of the five identified compounds using the percentage area integration values of the MS and PDA chromatograms of the EtOH and MeOH/DCM (1:1) extracts. a

receptor antagonist, provide only a symptomatic treatment.15 Hence, the development of potential drug targets and therapeutic agents is needed for effective AD treatment. Monsonia angustifolia (coded BP21) is traditionally used for food and cooked as an indigenous vegetable for daily meals in Tanzania.16 BP21 is also traditionally used in South Africa as a medicinal plant to treat erectile dysfunction. Recently, it is reported that BP21 has pro-sexual stimulatory effects in male rats.17 In addition, several other species of the Monsonia genus have been used for the treatment of dysentery, typhoid fever, intestinal hemorrhage, and diarrhea.18 However, there has been no report of BP21 related to the treatment of neurodegenerative diseases. In this study, we investigated the effect of BP21 extract on Aβ production and the spatial learning ability in the experimental models of AD. We found that the 60% ethanol (EtOH) extract of BP21 decreased Aβ levels in vitro. We also observed the recovering effect of the 60% EtOH extract of BP21 on memory impairment using the Morris water maze (MWM) test and novel object recognition test (NORT) in vivo. The 60% EtOH extract of BP21 significantly reduced the levels of insoluble Aβ42 in cerebral cortex and hippocampus. These results demonstrated that the 60% EtOH extract of BP21 improved spatial learning and memory of AD transgenic (TG) mice and decreased Aβ production. Next, we isolated and identified the following compounds from the BP21 extract: justicidin A, 5methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone. Among them, justicidin A was the most potent compound in decreasing Aβ levels in vitro. Thus, our data suggest that the 60% EtOH extract of BP21 has the potential to be developed as a treatment of AD. Furthermore, justicidin A may contribute, at least partially, to the Aβ reducing effect of the extract.

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acid for spectrometry (Sigma-Aldrich, St. Louis, MO, USA). Water was obtained from a Milli-Q RO system. Plant Materials. The aerial parts of BP21 were collected from January to February 2011 by botanist Hans Vahrmeijer from Limpopo province, Chuenespoort (GPS 24 10′559 S, 29 2′057 E), South Africa. Voucher specimen 39250002 was preserved at a herbarium of the South African National Biodiversity Institute in Pretoria. Spectroscopic Analyses. 13C NMR (125.7 MHz) and 1H NMR (500 MHz) spectroscopic data of the isolated compounds were obtained from the Unity Inova 500 MHz spectrometer (Varian, Palo Alto, CA, USA) with standard pulse sequences operating at 500 MHz for 1H and at 125 MHz for 13C NMR. The internal reference standard used was tetramethylsilane. Spectra were obtained from solutions in deuterium solvents. HPLC-MS. A Waters 2695 separation module linked to a Waters Acquity SQ detector was used for the analysis of plant extract and purified compounds. The column used was a Restek Ultra Biphenyl with 3 μm particle size and dimensions of 100 × 2.1 mm. The mass spectrometer settings were as follows: capillary voltage, 3 kV; cone voltage, 30 V; source temperature, 150 °C; desolvation temperature, 400 °C; desolvation gas flow, 600 L/h; and cone gas flow, 20 L/h. A binary solvent mixture was used consisting of water (eluent A) containing 10 mM formic acid (natural pH of 2.3) and acetonitrile (eluent B). The initial conditions were 95% A at a flow rate of 0.2 mL/ min and were maintained for 7 min, followed by a linear gradient to 60% A at 10 min, and ending at 60% A at 45 min. The conditions were kept constant for 1 min and then changed to the initial conditions. The PDA detector scanned between 200 and 500 nm (1.2 nm resolution). Preparation of BP21 Extract. Ground material (1 kg) of BP21 was extracted with 6 L of 60% EtOH by agitation for 24 h. The extract was then filtered through Merck no. 1 filter paper. The extraction of the residue was repeated twice more for an hour each time. The filtrates obtained were combined and evaporated to remove most of the ethanol. The resultant aqueous extract was freeze-dried to dryness, resulting in 121 g of powder (yield, 12.1%). Isolation and Analysis of Active Compounds. The compounds were isolated from the methanol and dichloromethane (MeOH/ DCM) extract [1:1 (v/v)]. A comparison was made between the chemical profiles of the 60% EtOH and MeOH/DCM (1:1) extracts, and the relative quantities of the five compounds determined in the 60% EtOH extract were compared to those of the MeOH/DCM (1:1) extract (Supporting Information Figure S1). Relative quantification was performed by comparing the integrated peak areas of the five identified compounds (Table 1). With regard to compound isolation from the dried and ground whole plant of BP21, 266 g was extracted with a 6 L mixture of MeOH/DCM (1:1) solvents for 4 h at room temperature with stirring and then filtered through Merck filter paper. The residue was washed with 3 L of MeOH/DCM (1:1) solvent followed by filtration. The

MATERIALS AND METHODS

Chemicals. Absolute EtOH (99.9%) supplied by Merck (Rahway, NJ, USA) was used for the purification of compounds. A solution of 60% ethanol extract was obtained by diluting 99.9% ethanol with purified water, as needed. All of the other reagents used were of analytical grade. HPLC grade solvents were purchased from Romil (Cambridge, UK) for analyses. The following reagents were used for LC-MS analysis: isopropyl alcohol Berdick & Jackson high-purity isopropyl alcohol (Honeywell Burdick & Jackson, Morristown, NJ, USA), acetonitrile 200 far UV (Romil, Cambridge, UK), and formic B

DOI: 10.1021/acs.jafc.6b04451 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry filtrates from both extractions were combined and evaporated under vacuum at a reduced pressure and a temperature of 40 °C. A greenishbrown extract was obtained after the solvent was completely removed, resulting in 27.3 g of extract (yield, 10.3%). Six grams of the obtained extract was subjected to repeated silica gel column chromatography (silica gel 60, Merck, 230−400 mesh) using a mixture of hexane and ethyl acetate at a ratio of 8.5:1.5 (v/v) as an elution solution to elute and to isolate the compounds from the extract. From BP21 MeOH/DCM (1:1) extract, 19.3 mg of justicidin A (white powder; yield of 0.3%), 35 mg of 5-methoxyjusticidin A (white powder; yield of 0.6%), 27 mg of chinensinaphthol (white powder; yield of 0.45%), 16 mg of retrochinensinaphthol methyl ether (yellow crystalline; yield of 0.26%), and 36 mg of succilactone (yellow crystalline; 0.6%) were obtained. In comparison, the yields of each compound from BP21 60% EtOH extract were 0.05, 0.19, 0.02, 0.03, and 0.1%, respectively. The structures of the compounds were elucidated with NMR analysis data and mass spectral analysis. Cell Culture. HeLa cells stably transfected with APP751 carrying the Swedish mutation (APPsw) were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum containing 100 units/mL penicillin, 100 μg/mL streptomycin, 260 μg/mL Zeocin, and 400 μg/ mL G418. Cell Viability Measurement. Cell viability was analyzed by an EZ-Cytox kit (Daeil Lab Co., Ltd., Seoul, Republic of Korea) according to the supplier’s instructions. Cells at 80% confluence in a 96-well plate were cultured for 8 h with the EtOH extract of BP21 or active compounds dissolved in DMSO. Then, cells were incubated with EZ-Cytox solution for 1 h at 37 °C. The absorbance at 450 nm was detected using a microplate reader. Aβ Peptide Assay. Cells at 80% confluence in a 35 mm dish were cultured for 8 h with the EtOH extract of BP21 or active compounds dissolved in DMSO in DMEM culture medium without serum. Control cells were treated similarly and incubated with a serum-free DMEM solution containing DMSO. The conditioned media were analyzed using a sandwich enzyme-linked immunosorbent assay (ELISA; Invitrogen, CA, USA) specific for Aβ42 or Aβ40 according to the supplier’s instructions. Animals and Drug Administration. The 3-month-old male APPsw TG mice [strain name, B6; SJL-Tg (APPSWE) 2576Kha; model no. 1349] used in this study were obtained from Taconic Biosciences (Hudson, NY, USA). This model was created by microinjecting the human APP695 gene, containing the doublemutation K670N and M671L, into B6SJLF2 zygotes using a hamster prion protein cosmid vector. The resultant mice from Founder Line 2576 were backcrossed to C57BL/6. All animals were housed in solidbottom cages with pellet food and water available ad libitum. The mice were also maintained on a 12/12 h light/dark cycle in a temperatureand humidity-controlled room (23 °C, 50%). The first set of TG and background mice were randomly grouped at the age of 9 months. A 60% EtOH extract of BP21 at doses of 100 and 250 mg/kg was administered daily per os until the end of all experiments. The second set of TG and background mice were randomly grouped at the age of 11 months. A 60% EtOH extract of BP21 at doses of 150, 250, and 350 mg/kg was administered daily per os until the end of all experiments. The other groups of mice served as controls and were administered 5% Tween 80−-saline solution during the drug administration period. The animal protocols used in this study were in accordance with and granted (Grant AP-2010KB001) by the Korea Institute of Science and Technology Animal Care and Ethics Committee. Morris Water Maze Test. At the age of 14 months, the mice were subjected to the MWM test, which is one of the behavioral tests for studying spatial learning and memory.19 The MWM consists of a circular pool (120 cm in diameter and 60 cm in depth) filled with water at 24−26 °C to a depth of 40 cm. The water was made opaque (white in color) using water-soluble nontoxic paint. A nonvisible escape platform, 8 cm in diameter, was submerged approximately 1 cm below the water surface in the center of the designated target quadrant of the pool. The two phases of the MWM tests, acquisition and retention, were conducted for 7 consecutive days. The acquisition

phase was conducted by putting each TG mouse in the pool to swim for a total of three trials per day during the first 6 days, with a 1 h intertrial interval. Mice were given a maximum of 90 s to find the platform and had to remain on the platform for at least 5 s. If the mouse was unable to find the platform within the 90 s time frame, it was placed directly on the platform for 5 s and then returned to its cage. For each trial, the mouse was randomly placed under one of the four visual cues. During the acquisition phase, the amount of time required for the mouse to find the hidden platform (escape latency) was measured. For those mice that did not find the hidden platform in the allotted time, a value of 90 s was assumed. The retention phase of the MWM occurs on the day immediately after the last day of the acquisition phase. During the retention phase, the platform was removed and mice were given 120 s to freely explore the pool. The total duration of time spent in the target quadrant that had contained the escape platform during the acquisition phase was measured. After the test, mice were removed from the pool and returned to their cages. Novel Object Recognition Test. After the MWM test, mice were rested for 1 week, and then a NORT was conducted for studying the spontaneous tendency of mice to explore a novel object more often than a familiar one.20 A plastic chamber (50 cm × 50 cm × 50 cm) with 10-cm-spaced grids at the bottom was used. The NORT procedure consisted of three different phases: habituation for 3 days, acquisition for 1 day, and retention for 1 day. In the habituation phase, mice were individually subjected to a single 10 min familiarization session during which they were introduced to an empty arena. On the fourth day (acquisition phase), mice were subjected to a single 10 min exploring session, during which two floor-fixed objects (A and B) were placed in a symmetric position from the center of the arena with enough space between them and the walls. Both plastic objects were the same in color, smell, shape, and size. Mice were allowed to explore the objects freely in the arena. On the fifth day (retention phase), mice were subjected to a single 5 min exploration session. During this session, mice were placed in the arena that contained two objects, A and C. Object C was a novel object with a different color, smell, and shape when compared to object B of the acquisition phase. The memory index for each mouse was expressed as a ratio of the amount of time spent exploring the novel object C, (TC × 100)/(TA + TC), where TA and TC were the time spent on exploring objects A and C during the 5 min period, respectively. In Vivo Insoluble Aβ42 Detection. For in vivo detection of insoluble Aβ42 peptides in the brain, the TG mice that had undergone behavior tests were anesthetized and decapitated at the age of 15 months. The cerebral cortex and hippocampus was dissected from the exposed brains, and the tissue samples were stored at −80 °C until use. The brain tissue samples were homogenized in Tris-buffered saline solution (20 mM Tris; 137 mM NaCl; pH 7.4) containing a complete protease inhibitors tablet (Sigma, St. Louis, MO, USA). The extraction ratio (brain tissue/Tris-buffered saline) was set at 1:10 (w/v), and then 200 μL of tissue homogenates was added to a 400 μL of formic acid (minimum 95%), sonicated, and centrifuged at 100,000g for 1 h at 4 °C. Subsequently, 210 μL of the sample was neutralized in 4 mL of formic acid neutralization buffer (1 M Tris base, 0.5 M Na2HPO4, 0.05% NaN3). Insoluble Aβ42 levels were determined using the same sandwich ELISA kit used for the in vitro detection. Statistical Analysis. All of the results are presented as the mean ± SEM. The overall significance of experimental results was examined by Levene’s test for variance homogeneity. A one-way analysis of variance was then conducted along with a two-tailed Dunnet t test. Differences between the groups were considered significant if p < 0.05 with the appropriate Bonferroni correction for multiple comparisons.

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RESULTS AND DISCUSSION BP21 Extract Decreased the Levels of Secreted Aβ42 and Aβ40 in APPsw-Transfected HeLa Cells. We tested the effect of the 60% EtOH extract of BP21 on Aβ42 and Aβ40 production in HeLa cells stably transfected with APPsw. Cells were incubated with 1, 5, 10, 25, and 50 μg/mL of BP21 extract for 8 h, and the Aβ42 and Aβ40 levels from the conditioned C

DOI: 10.1021/acs.jafc.6b04451 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry media were measured by using specific ELISA kits. The secreted level of Aβ42 was significantly decreased by the 60% EtOH extract of BP21 in a dose-dependent manner (Figure 1A;

Figure 2. Effect of BP21 extract on the Morris water maze test. The 60% EtOH extract of BP21 (100 mg/kg/day; n = 5 or 250 mg/kg/day; n = 7) or saline was administered orally to 9-month-old TG mice (control; n = 9) or normal mice (background; n = 8) for 6 consecutive months and 1 h before the first trial of each acquisition day. (A) The acquisition phase consisted of 6 consecutive days in which each TG mouse accomplished three trials with a 1 h intertrial interval. The amount of time taken to find the hidden platform (escape latency) was measured during the acquisition phase. Treatment with the 60% EtOH extract (250 mg/kg) of BP21 significantly enhanced the escape latency. (B) The retention phase was followed the last day of the acquisition phase. The platform was removed, and the mice were given 120 s to explore the pool. The time spent in the target quadrant that had contained the escape platform during the acquisition phase was measured. Treatment with the 60% EtOH extract (250 mg/kg) increased the time spent in the target quadrant, compared with the control group. (∗∗) P < 0.01 from the background group. (#) P < 0.05 from the TG control group.

Figure 1. Effect of BP21 extract on the levels of Aβ42 and Aβ40 in APPsw-transfected cells. APPsw-transfected HeLa cells were incubated with the indicated concentration of the 60% EtOH extract of BP21 for 8 h. The media were collected and analyzed for Aβ levels by using sandwich ELISA. (A) The level of Aβ42 was decreased by the 60% EtOH extract of BP21 at 5, 10, 25, and 50 μg/mL (n = 4). (B) The level of Aβ40 was decreased by the 60% EtOH extract of BP21 at 5, 10, 25, and 50 μg/mL (n = 4). (∗) P < 0.05; (∗∗) P < 0.01; (∗∗∗) P < 0.001.

n = 4). When cells were incubated with 25 and 50 μg/mL of the 60% EtOH extract of BP21, the level of Aβ42 was significantly decreased by 46 ± 1.2 and 25 ± 2.9%, respectively, compared to the control. In addition, the secreted level of Aβ40 was also significantly decreased by the BP21 60% EtOH extract in a dose-dependent manner (Figure 1B; n = 4). In particular, 25 and 50 μg/mL of the 60% EtOH extract of BP21 significantly decreased the levels of Aβ40 by 30.3 ± 2.4 and 14.7 ± 1.7%, respectively. Furthermore, the 60% EtOH extract of BP21 did not influence cell viability (data not shown). These results suggest that the EtOH extract of BP21 decreases the secreted levels of Aβ42 and Aβ40, without inducing cell toxicity. Long-Term Treatment with BP21 Extract Improved the MWM Test Performance. Our initial finding on the Aβ reducing effect of BP21 EtOH extract in vitro led us to observe its effect in AD TG mice in the aspects of brain Aβ reduction and recovery of memory function. To assess the effect of longterm treatment with the BP21 extract on memory capacity of TG mice, we tested the first set of TG mice using the MWM. The MWM is one of the behavioral tests for studying hippocampal-dependent spatial learning and memory.19 We divided TG mice into four groups that received either control (n = 9), BP21 60% EtOH extract 100 mg/kg (n = 5), or BP21 60% EtOH extract 250 mg/kg (n = 7). At the age of 14 months, the learning trials were conducted over 6 days. Spatial learning was measured using escape latency, which is defined as the time required to find the hidden platform. As shown in Figure 2A, the non-TG background mouse group exhibited a significant decrease in escape latency from the first day (90.1 ± 0 s) to the sixth day (52.2 ± 7.7 s) of the acquisition phase. In contrast, the TG mice (control) showed no significant difference in finding the escape platform from the first day (86.6 ± 6.5 s) to the sixth day (87.4 ± 1.8 s), exhibiting significantly impaired memory function. Long-term treatment with the 60% EtOH extract of BP21 showed improved memory function by reducing escape latency, starting from the third acquisition day. The most significant reduction in escape latency was observed on the last acquisition day in both the 100 and 250 mg/kg 60% EtOH extracts of BP21-treated groups to 71.6 ±

4.3 s and 59 ± 7.4 s, respectively. Following the acquisition phase, we tested the retention of spatial memory on the seventh day. As shown in Figure 2B, the duration of time spent in the target quadrant that contained the escape platform was significantly reduced in the TG mouse group (control; 19.0 ± 3.7 s), compared with that in the non-TG mouse group (background; 37.2 ± 3.1 s). However, the group treated with 250 mg/kg of 60% EtOH extract of BP21 showed a significant increase in the time in the target quadrant (30.4 ± 2.8 s) compared to the control group, exhibiting an improved spatial memory ability of TG mice. These results indicate that longterm oral treatment with 60% EtOH extract of BP21 enhances the memory function lost in TG mice. Long-Term Treatment with the BP21 Extract Improved Object Memory Function in the Novel Object Recognition Test. We further assessed the effect of the BP21 extract on recognition memory using a NORT. The NORT is a highly validated test for recognition memory.20 We found that the non-TG mice (background) more frequently interacted with a novel object than a familiar object by the novel object preference ratio of 85% (Figure 3). In contrast, TG mice (control) showed a decreased preference ratio of 57%, indicating an impairment in recognition memory in TG mice. Long-term treatment with 250 mg/kg of 60% EtOH extract of BP21 exhibited a significant recovery in the preference ratio to a normal range of 82%. Taken together with MWM test results, it appears that the 60% EtOH extract of BP21 significantly ameliorates behavioral deficits in TG mice after a long-term oral treatment. Long-Term Treatment with BP21 Extract Decreased the Level of Aβ42 in the TG Mouse Brains. The amelioration of memory functions in AD TG mice might reflect a decrease or attenuation of brain damage commonly observed in AD pathology. One of the most relevant factors to neuronal damage in AD is accumulation of Aβ. To examine D

DOI: 10.1021/acs.jafc.6b04451 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(control) also markedly increased to 3.9 ± 0.8 μg/mL, compared with that in the non-TG mouse group (background) (Figure 4B). The BP21 60% EtOH extract significantly reduced the level of insoluble Aβ42 in the hippocampus, compared with that in the control. Treatment with 100 and 250 mg/kg of 60% EtOH extract of BP21 reduced the levels of Aβ42 to 2.0 ± 0.3 and 1.7 ± 0.5 μg/mL, respectively. Taken together with the obvious improvement of cognitive function, the decrease of insoluble Aβ42 levels in mouse brains shows the beneficial effects of the 60% EtOH extract on an AD mouse model. The 60% EtOH Extract of BP21 Ameliorated Spatial Learning in TG Mice. On the basis of successful results of BP21 60% EtOH extract with long-term treatment, we continued to test and confirm if increased dosage over a shorter treatment period would also show beneficial effects in AD TG mice model. TG mice were fed 150, 250, and 350 mg/ kg of 60% EtOH extract of the BP21 per os for 4 months. Figure 5A shows the results of MWM acquisition training. The non-TG background mouse group exhibited a significantly decreased escape latency from the first day (81.2 ± 4.4 s) to the sixth day (28.9 ± 4.4 s) of acquisition. As expected, TG mice (control) showed no significant difference in the escape latency from the first day (90.1 ± 0 s) to the sixth day (75.9 ± 7.9 s). We found that BP21 extract-treated groups showed a tendency for reduced escape latency on the last day of MWM acquisition training. TG mice treated with 350 mg/kg of 60% EtOH extract of BP21 showed the most significant improvement (55.1 ± 13 s). The duration of the target quadrant on the seventh day of the MWM test was significantly reduced in the TG mouse group (control; 19 ± 3.7 s) compared to that in the non-TG mouse group (background; 37.2 ± 3.1 s) (Figure 5B). With the tendency of increased duration in the target quadrant with 60% EtOH extract of BP21 treatment, significance was observed in the 250 mg/kg of BP21 extract treated group (30.4 ± 2.8 s). We further assessed the effect of the BP21 extract on recognition memory by using a NORT. As shown in Figure 5C, the non-TG mice (background) spent over 76% of the exploring time on a novel object, whereas TG mice (control) spent only 56% of the time on a novel object. We found that treatment with 250 and 350 mg/kg of 60% EtOH extract of BP21 significantly increased the time, up to 70.2 and 76.7%, respectively. These results indicate that the 60% EtOH extract of BP21 ameliorated the behavioral deficits of AD TG mice over a shorter drug treatment duration. Overall, the 60% EtOH extract of BP21 has a recovering effect on memory impairment of AD TG mice while reducing the Aβ level in the brain, and a dosage of 250 mg/kg was enough to induce those significant changes. On the basis of these observations, we hypothesized that these beneficial effects of BP21 EtOH extract could be affected, at least partially, by the reduction of Aβ in the brain. Therefore, further investigation was conducted to find the active compound candidates based on 60% EtOH extract of BP21. Chemical Structure Elucidation of Isolated Compounds. From multiple wide-range in vitro screening tests for active compounds with Aβ reducing effect in BP21 60% EtOH extract, many candidate compounds from the major constituent group have failed to show such an effect (data not shown). However, among the short-listed compounds, justicidin A and 5-methoxyjusticidin A, which share the aryl naphthalene structure, exhibited strong Aβ reducing effect starting from low-concentration treatment. In extension to such

Figure 3. Effect of BP21 extract on novel object recognition test. The 60% EtOH extract of BP21 (100 mg/kg/day; n = 5 or 250 mg/kg/day; n = 7) or saline (control; n = 9) was administered orally to 9-monthold TG mice or normal mice (background; n = 8) for 6 consecutive months and 1 h before the acquisition phase. Mice were placed into a square-shaped arena and were accustomed to two same (familiar) objects for 10 min. On the following day, mice were placed within the same arena, and one familiar object and one novel object were placed into the arena and the mice allowed to explore freely for 5 min. The accumulated exploring time for each object was measured, and the memory index was calculated according to the following equation: memory index (%) = (exploring time on novel object/total exploring time for both objects) × 100. Treatment with 250 mg/kg 60% EtOH extract of BP21 significantly improved the ratio of the amount of time spent exploring the novel object. (∗∗∗) P < 0.001 from the background group. (##) P < 0.01 from the TG control group.

whether long-term treatment with the BP21 extract is effective for reducing Aβ42 production in the brain of TG mice, we quantitated the level of insoluble Aβ42 in both the cerebral cortex and the hippocampus in mice aged 15 months. The level of insoluble Aβ42 in the cerebral cortex of TG mice (control) markedly increased to 4.3 ± 0.01 μg/mL from insignificant background level in the non-TG mouse group (background) (Figure 4A). Groups treated with the 60% EtOH extract of BP21 exhibited a tendency for reducing insoluble Aβ42 levels in the cerebral cortex, which was most significant in the group treated with 250 mg/kg of 60% EtOH extract of BP21. The level of insoluble Aβ42 in the hippocampus of TG mice

Figure 4. Effect of BP21 extract on the level of insoluble Aβ42 in the mouse cerebral cortex and hippocampus. Brain tissue of mice was collected after behavioral tests at 15 months of age. The cerebral cortex and hippocampus samples were homogenized at 10% (w/v) in tissue homogenization buffer. The tissue homogenates were added to formic acid, centrifuged at 100,000g for 1 h at 4 °C, and then neutralized with formic acid neutralization buffer. The level of insoluble Aβ42 was measured using a sandwich ELISA kit. The 60% EtOH extract of BP21 significantly reduced the level of insoluble Aβ42 in cerebral cortex (A) and hippocampus (B). (∗∗) P < 0.01; (∗∗∗) P < 0.001 from the background group. (#) P < 0.05 from the TG control group. E

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To confirm the established structures, the chemical shift data were compared to data from previously identified and known compounds online or in structural databases. The structures of the selected five compounds were confirmed as aryl naphthalene lignans, which are biosynthetically formed by fusing at least two phenyl propanyl (C6−C3) units (Figure 6). Compound 1 has been identified as justicidin A with m/z 394 [M]+ (C22H18O7). This compound is one of the most commonly known lignans found in plants.21,22 Compound 2 was unambiguously identified as 5-methoxyjusticidin A, which was previously isolated from the plant Justicia procumbens, with a molecular formula C23H20O8 and a corresponding molecular ion at m/z 424 [M]+.23 Compound 3 is commonly known as chinensinaphthol and was previously isolated from J. procumbens; it reportedly has antiplatelet properties.24 A mass charge of m/z 380 was obtained for this compound, consistent with the proposed molecular formula C21H16O7. Compound 4 (C22H18O7) was elucidated as retrochinesinaphthol methyl ether in conjunction with spectral data, and a molecular ion [M]+ of m/z 394 was obtained.25 Compound 5 (C21H20O6) was found to have m/z 368 [M]+ and was assigned the name suchilactone, based on literature and NMR spectral data comparison using authenticated published data.26,27 The NMR data from five isolated compounds are shown in Supporting Information Tables S1−S5. Isolated Compounds from BP21 Extract Decreased the Levels of Aβ40 and Aβ42 in APPsw-Transfected HeLa Cells. The five isolated compounds, justicidin A, 5methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone, were treated with various concentrations in APPsw-transfected HeLa cells for 8 h. The levels of Aβ42 and Aβ40 from the conditioned media were measured using specific ELISA kits. Both Aβ42 and Aβ40 levels were potently decreased by justicidin A in a dose-dependent manner (Figure 7A; n = 4). At 1.25 μM, justicidin A significantly decreased the levels of Aβ42 and Aβ40 by 82.2 ± 2.3 and 44.9 ± 0.3%, respectively. In addition, the level of Aβ42 was significantly decreased by 51.4 ± 1.4 and 75.6 ± 1.7% at 1.2 and 11.8 μM 5-methoxyjusticidin A, respectively (Figure 7B; open bars; n = 4). The level of Aβ40 was also significantly decreased by 10.5 ± 1.3 and 58.8 ± 0.9% at 1.2 and 11.8 μM 5methoxyjusticidin A, respectively (Figure 7B; solid bars; n = 4). Furthermore, we have confirmed that both justicidin A and 5methoxyjusticidin A did not influence cell viability (Supporting Information Figure S2). Chinensinaphthol (Figure 7C; n = 2) and suchilactone (Figure 7E; n = 2) showed only a trend toward decreasing Aβ42 levels, whereas retrochinensinaphthol methyl ether (Figure 7D; n = 2) led to increased Aβ42 levels without statistical significance. With the current observation, it will be reasonable to expect that justicidin A and 5methoxyjusticidin A will be the two active compounds that could possibly affect the Aβ reducing effect of BP21 EtOH extract. We further observed the composition status of active components in the 60% EtOH extract using HPLC analysis (Supporting Information Figure S3). The area under the peaks including the selected five compounds as well as several other justicidin-related compounds in the boxed region was calculated as percentage. The BP21 60% EtOH extract appeared to contain justidicins in the quantity of 0.8%. Currently illustrated results may partially explain the beneficial effects of BP21 60% EtOH extract on cognitive function improvement and Aβ reduction in AD TG mice.

Figure 5. Effect of short-term treatment of BP21 extract on memory impairment. The 60% EtOH extract of BP21 (150 mg/kg/day; n = 6, 250 mg/kg/day; n = 5 or 350 mg/kg/day; n = 6), or saline was administered orally to 11-month-old TG mice (control; n = 6) or normal mice (background; n = 10) for 4 months and 1 h before the first trial of each acquisition day. (A) Treatment with the 60% EtOH extract (350 mg/kg) of BP21 significantly enhanced the escape latency. (B) Treatment with the 60% EtOH extract (250 mg/kg) of BP21 increased the time spent in the target quadrant, compared to that of the control group. (C) Treatment with 250 and 350 mg/kg of 60% EtOH of BP21 extract significantly rescued the ratio of the time spent exploring the novel object. (∗∗) P < 0.01; (∗∗∗) P < 0.001 from the background group. (#) P < 0.05; (##) P < 0.01 from the TG control group.

a finding, we focused on additional discovery of active compounds with structure similar to that of justicidin A. The hydrophobic justicidin A and other compounds were isolated from the BP21 MeOH/DCM (1:1) extract for maximum efficiency, and the structures of the isolated metabolites were elucidated using spectral analysis. The 1H and 13C NMR spectral data provided the first stage in the characterization of the compounds. Wherever necessary, advanced homonuclear and heteronuclear two-dimensional NMR methods (COSY, HMQC, and HMBC) were applied to achieve complete assignments of the 1H and 13C correlations in the isolated compounds. From the proton-decoupled DEPT pulse sequence subspectra, the protonated carbons were assigned according to their multiplicity. F

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Figure 6. Chemical structures of active compounds: compound 1, justicidin A, 9-(1′,3′-benzodioxol-5′-yl)-4,6,7-trimethoxynaphtho[2,3-c]furan1(3H)-one; compound 2, 5-methoxyjusticidin A, 9-(1′,3′-benzodioxol-5′-yl)-4,5,6,7-tetramethoxynaphtho[2,3-c]furan-1(3H)-one; compound 3, chinensinaphthol, 9-(3′,4′-dimethoxyphenyl)-4-hydroxy-6,7-methylenedioxynaphtho[2,3-c]furan-1(3H)-one; compound 4, retrochinensinaphthol methyl ether, 4-(3′,4′-dimethoxyphenyl)-9-methoxy-6,7-methylenedioxynaphtho[2,3-c]furan-1(3H)-one; compound 5, suchilactone, 3-(1′,3′benzodioxol-5′-ylmethylene)-4-(3″,4″-dimethoxybenzyl)dihydrofuran-2(5H)-one.

learning ability in vivo, as we did not isolate all of the compounds present in BP21 extract. Furthermore, in the aspect of natural product research, the active compounds in extract or fraction composition often get help in drug delivery, absorption rate, metabolic processes, etc., by other minor components in the mixture. We assume that justicidin A and 5-methoxyjusticidin A may be affected by the other components in similar ways in vivo. Therefore, further research on the effect of active compounds found in this study should be conducted regarding not only the sole effect of the compound itself but also collaborative effects in coordination with other components found in the extract. Previously, we reported the neuroprotective effect of justicidin A on the pathogenesis of AD.28 Justicidin A inhibited tau hyperphosphorylation in Aβ25−35-induced SH-SY5Y neuroblastoma cell. However, there is no report that other active compounds from BP21 have activities against AD in vivo or in vitro. Therefore, further intensive study on justicidin A and the extract containing it will be required to identify the underlying molecular mechanisms by which it decreases Aβ production to explain its efficacy in alleviating AD pathogenesis.

Figure 7. Effect of the isolated compounds from the BP21 extract on Aβ40 and Aβ42 production in APPsw-transfected cells. APPswtransfected cells were incubated with the indicated concentration of justicidin A, 5-methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone for 8 h. The media were collected and analyzed for Aβ levels by using sandwich ELISA. (A) The levels of both Aβ42 (open bars; n = 4) and Aβ40 (solid bars; n = 4) were decreased by justicidin A in a dose-dependent manner. (B) The levels of both Aβ42 (open bars; n = 4) and Aβ40 (solid bars; n = 4) were decreased by 5-methoxyjusticidin A. (C) Chinensinaphthol showed a trend toward decreasing Aβ42 level (n = 2). (D) Retrochinensinaphthol methyl ether increased the level of Aβ42 (n = 2). (E) Suchilactone showed a trend toward decreasing Aβ42 levels (n = 2). (∗) P < 0.05; (∗∗) P < 0.01; (∗∗∗) P < 0.001.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04451. HPLC-MS and HPLC-UV chromatograms of the BP21 MeOH/DCM [1:1] extract and the 60% EtOH extract; lack of effect of justicidin and 5-methoxyjusticidin A on cell viability; HPLC analysis of BP21 extracts; NMR data of compounds 1 (justicidin A), 2 (5-methoxyjusticidin A), 3 (chinensinaphthol), 4 (retrochinensinaphthol methyl ester), and 5 (suchilactone) in CDCl3; (PDF)

However, there remains the possibility that other active compounds may also influence the Aβ production and spatial G

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AUTHOR INFORMATION

Corresponding Authors

*(H.O.Y.) Phone: +82 33 650 3501. Fax: +82 33 650 3529. Email: [email protected]. *(G.F.) Phone: +27 12 841 3815. Fax: +27 12 841 3561. Email: [email protected]. ORCID

Hyun Ok Yang: 0000-0003-1604-0843 Author Contributions ∥

Y.S.C. and J.K. contributed equally to this work.

Funding

This work was funded and supported by the Bio-Synergy Research Project (NRF-2012M3A9C4048793) and the Bio & Medical Technology Development Program (NRF2015M3A9A5030735) of the Ministry of Science, ICT, and Future Planning through the National Research Foundation, Republic of Korea to H.O.Y. Also, this work was supported by NRF (2013R1A1A2006475) to S.C. and through a Parliamentary Grant, South Africa (2014GWDMS240117). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED Aβ, β-amyloid; AD, Alzheimer’s disease; ADAM, A disintegrin and metalloproteinase; APP, β-amyloid precursor protein; BP21, Monsonia angustifolia; MWM, Morris water maze; NORT, novel object recognition test; TG, transgenic

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