Bioassay-Guided Isolated Compounds from Morinda officinalis Inhibit Alzheimer’s Disease Pathologies

Due to the side effects of synthetic drugs, the therapeutic potential of natural products for Alzheimer’s disease (AD) has gained interest. Morinda officinalis has demonstrated inhibitory effects on geriatric diseases, such as bone loss and osteoporosis. However, although AD is a geriatric disease, M. officinalis has not been evaluated in an AD bioassay. Therefore, M. officinalis extracts and fractions were tested for AD-related activity, including inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and advanced glycation end-product (AGE) formation. A bioassay-guided approach led to isolation of 10 active compounds, eight anthraquinones (1–8), one coumarin (9), and one phytosterol (10), from n-hexane and ethyl acetate fractions of M. officinalis. The five anthraquinones (4–8) were stronger inhibitors of AChE than were other compounds. Compounds 3 and 9 were good inhibitors of BChE, and compounds 3 and 8 were good inhibitors of BACE1. Compounds 1–5 and 7–9 were more active than the positive control in inhibiting AGE formation. In addition, we first suggested a structure-activity relationship by which anthraquinones inhibit AChE and BACE1. Our findings demonstrate the preventive and therapeutic efficacy of M. officinalis for AD and its potential use as a natural alternative medicine.

Data are presented as the mean ± S.D. (n = 3); a IC 50 calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity; b Berberine was used as a positive control of AChE and BChE inhibition.; c AG was used as a positive control of inhibition of AGE formation; d Quercetin was used as a positive control of BACE1 inhibition; e ND was not detectable; * indicates a significant difference from control; * p < 0.05, ** p < 0.005, *** p < 0.001

Discussion
In recent years, the aging society and increasing life span have increased the number of people over 65 years old worldwide. As a result, degenerative and geriatric diseases are increasing. Dementia, a major symptom of cognitive disorders, is a significant social problem [36]. While dementia can result from degenerative dementia, senile dementia, Parkinson's disease, and AD, AD is the most common, accounting for 50% to 60% of all dementia [37]. M. officinalis has already been demonstrated to inhibit geriatric diseases such as bone loss and osteoporosis. Although AD is a geriatric disease, M. officinalis has not been evaluated in an AD bioassay. Therefore, we aimed to assess whether M. officinalis has the potential to treat AD by inhibit AChE, BChE, BACE1, and AGE formation.
M. officinalis extracts and fractions were investigated for their ability to inhibit AChE, BChE, BACE1, and AGE formation. The M. officinalis extracts were good inhibitors of AChE, BChE, and BACE1. The extracts inhibited BACE1 more strongly than did the other fractions. The Hx fraction was a stronger inhibitor in all assays. The Hx fraction inhibited AChE, BChE, and AGE formation significantly more than the other fractions. The EA fraction mildly inhibited AChE, BACE1, and AGE formation. In contrast, the BuOH and water fractions had no, or slight, activity in all assays. These results demonstrated that the potential of M. officinalis extracts to prevent AD was derived from the Hx and EA fractions.
Therefore, we conducted bioassay-guided isolation from the Hx and EA fractions. We isolated bioactive compounds, including eight anthraquinones (1-8), one coumarin (9), and one phytosterol (10). The isolated compounds 1-10 were investigated for inhibition of AChE, BChE, BACE1, and AGE formation. Previous literatures studied AD activities of various natural products, for examples, cholinesterase activities of flavonoid isolated from Kaempferia parviflora, Maclura pomifera, essential oils of Salvia species, and their crude extracts [35,38,39]. When we compared previous articles with our data, it could know that anthraquinones had more potential than the natural products kind of flavonoids and fatty acids. Taken together, our study was significant to have accessed the anti-AD activities of anthraquinones.
Compounds 4-8 were stronger AChE inhibitors than other compounds. Of these, compound 5 was the most active. Furthermore, we uncovered the following relationships between the anthraquinone structure and AChE inhibitory activity: (1) anthraquinones with no substituent on C-1 (compounds 4 and 5) were more active than those with a substituent in C-1 (compounds 1-3 and 6-8); (2) anthraquinone with a substituted methyl group on C-2 (compounds 6 and 8) were more active than those with a methoxy group (compounds 2 and 3); (3) anthraquinones with a substituent on C-3 (compounds 2 and 4-8) had stronger activity than those without (compounds 1 and 3); (4) anthraquinones with a hydroxy group at C-3 (compounds 5 and 8) were more active than those with a methoxy group (compounds 4 and 6); and (5) the anthraquinone with no hydroxy group was a minor inhibitor (compound 3).
Compound 9 significantly inhibited BChE, and compound 3 slightly inhibited AChE, making them the most active among the isolated anthraquinones. According to bioassay-guided isolation, the EA fraction also showed low potential, because most anthraquinones isolated from the EA fraction had weak activity. The Hx fraction was the most active because compound 3, a good inhibitor, was isolated from Hx fraction.
Finally, compounds 1-5, 7, and 8 were stronger inhibitors of AGE formation than AG, the positive control. Compound 9 showed the best activity. Previous studies have indicated that scopoletin (9) is a remarkable inhibitor of AGE formation [40]. Our results indicated that anthraquinones with only one substituent (compound 3) were the most effective, anthraquinones with a hydroxy group (compounds 5 and 8) had more activity than those with other substituents (compounds 4 and 6), and anthraquinones with a methoxy group (compound 2) were stronger inhibitors than those with a methyl group (compound 6).
In conclusion, this study used bioassay-guided isolation to identify 10 compounds from M. officinalis. The isolated compounds inhibited AChE, BChE, BACE1, and AGE formation, which are related to AD. In addition, we suggested a structure-activity relationship for AChE and BACE1 inhibition by anthraquinones. These results demonstrated that M. officinalis root extracts were therapeutic and may be a natural medicine for treating AD.

Extraction, Fractionation, and Isolation of M. officinalis
Dried and powdered M. officinalis roots (3.9 kg) were extracted in MeOH (20 L × 3) at room temperature. The filtrate was concentrated to dryness (613.4 g) in vacuo; suspended in water (H 2 O); and partitioned in Hx, EA, and BuOH depending on solvent polarity. The result yielded Hx (3.84 g), EA (7.23 g), BuOH (192.81 g), and water (270.42 g) fractions. Among these three fractions, the Hx and EA fractions showed the most potent activities in the four anti-AD model assays. Therefore, we executed isolation from Hx and EA fractions.

Measurement of ChE Inhibitory Activities
ChE activity was detected by AChE-or BChE-mediated hydrolysis of DTNB for 15 min to form thiocholine and the yellow 5-thio-2-nitrobenzoate anion. The result was quantified by measuring the absorbance 412 nm. The assay mixture contained 0.1 M potassium phosphate buffer (pH 7.8), 0.3 U/mL AChE or BChE, 0.5 mM DTNB, 0.6 mM ACh or BCh, and the sample for a total volume of 0.2 mL. All tested samples were dissolved in 10% DMSO at five different final concentrations (10-500 µg/mL for extracts and fractions or 10-500 µM for isolated constituents). The reaction was performed in a 96-well plate. Berberine, a typical ChE inhibitor, was used as a positive control [16]. Inhibitory activity was calculated with the following formula: (Ac − As/Ac) × 100, where Ac is the change in absorbance for the control after 15 min and As is the change in absorbance for the sample after 15 min.

Measurement of BACE1 Inhibition
BACE1 inhibition was measured with a commercially available spectrophotometric method according to the manufacturer's recommended protocol. The assay mixture contained 50 mM sodium acetate buffer (pH 4.5), 1.0 U/mL BACE1, substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM ammonium bicarbonate), and sample. All tested samples were dissolved in 10% DMSO at five different final concentrations (2.5-1250 µg/mL for extracts and fractions or 2.5-1250 µM for isolated constituents). The reaction was incubated for 60 min at room temperature in the dark. BACE1 activity was determined by measuring the proteolysis of two fluorophores (Rh-EVNLDAEFK-Quencher) to form a fluorescent donor (Rh-EVNL) with an excitation of 545 nm and emission of 585 nm in a black 96-well plate. Quercetin, a typical BACE1 inhibitor, was used as a positive control [16,26,27]. Inhibition was calculated with the following formula: (Ac − As/Ac) × 100, where Ac is the change in fluorescence for the control after 60 min, and As is the change in fluorescence for the sample after 60 min.

Measurement of Inhibition of AGE Formation
Inhibition of AGE formation was measured with a spectrophotometric method developed previously [34]. All tested samples were dissolved in 10% DMSO at five different final concentrations (10-500 µg/mL for extracts and fractions or 10-500 µM for isolated constituents). The assay mixture contained bovine serum albumin (10 mg/mL), 50 mM phosphate buffer (pH 7.4) with 0.02% sodium azide, and 0.4 M fructose and glucose. The reaction was incubated at 60 • C for 2 days. After incubating, fluorescence was measured at an excitation wavelength of 350 nm and emission of 450 nm in a black 96-well plate. Aminoguanidine (AG), a typical inhibitor of AGE formation, was used as a positive control. The inhibitory activity was calculated with the following formula: (Ac − As/Ac) × 100, where Ac is the fluorescence of the control, and As is the fluorescence of the sample.

Statistical Analysis
All assays were performed in triplicate. Data are presented as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA. Data were considered statistically significant at p < 0.05.