RESEARCH PAPER

Effects of solvent fractions of Allomyrina dichotomalarvae through the inhibition of in vitro BACE1 andβ-amyloid(25–35)-induced toxicity in ratpheochromocytoma PC12 cellsMinji KIM1, Kumju YOUN1, Eun-Young YUN2, Jae-Sam HWANG2, Mok-Ryeon AHN1,Woo-Sik JEONG3 and Mira JUN1

1 Department of Food Science and Nutrition, Dong-A University, Busan, Korea2 Department of Agricultural Biology, National Academy of Agricultural Science RDA, Suwon, Korea3 Department of Food & Life Sciences, Inje University, Gimhae, Korea

Correspondence

Mira Jun, Department of Food Science andNutrition, Dong-A University, Hadan 2-dong,Saha-gu, Busan 604-714, Korea.Email: mjun@dau.ac.kr

Received 9 July 2013;accepted 28 November 2013.

doi: 10.1111/1748-5967.12046

Abstract

Amyloid-β peptide (Aβ) generation initiated by β-site amyloid precursor proteincleaving enzyme 1 BACE1 is a critical cause of Alzheimer’s disease. In the courseof our ongoing investigation of natural anti-dementia resources, the ethyl acetate(EtOAc) fraction exerted strong BACE1-specific inhibition with the half maximalinhibitory concentration (IC50) value of 9.2 × 10−5 μg/mL. Furthermore, Aβ(25–35)-induced cell death was predominantly prevented by the EtOAc fraction ofAllomyrina dichotoma larvae through diminishing of cellular oxidative stress andattenuating apoptosis by inhibiting caspase-3 activity. Taken together, the presentstudy demonstrated that A. dichotoma larvae possess novel neuroprotective proper-ties not only via the selective and specific inhibition of BACE1 activity but alsothrough the alleviation of Aβ(25–35)-induced toxicity, which may raise the pos-sibility of therapeutic application of A. dichotoma larvae for preventing and/ortreating dementia.

Key words: Allomyrina dichotoma larvae, Alzheimer’s disease (AD), amyloid β peptide (Aβ),β-secretase (BACE1)

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerativedisorder considered to be the most common cause ofdementia. The main pathological characteristics in AD areneurofibrillary tangles (NFT) and senile plaques composedof insoluble amyloid-β peptide (Aβ) (Glenner & Wong 1984;Beckman et al. 2006). Aβ itself is neurotoxic and abnor-mally excessive generation of Aβ leads to the formationof reactive oxygen species (ROS) and nitric oxide (NO) ,neuroinflammation and apoptosis, which causes more Aβgeneration, neuronal dysfunction and, ultimately, cell death(Loo et al. 1993; Hensley et al. 1994; Jang & Surh 2005).

Aβ peptides are proteolytically generated from the con-secutive cleavage of amyloid precursor protein (APP) by

β- and γ-secretases (Vassar 2002). This action is limitedwith processing by sequential α-secretase (TACE) and γ-secretase generating soluble and harmless P3 peptides thatare not involved in AD development (Holloway et al. 2009).β-Site amyloid precursor protein cleaving enzyme 1(BACE1) is proven to be the vital rate-limiting enzyme inthe formation of Aβ, a critical factor in AD (Cole & Vassar2007). Inhibition of BACE1 activity leads to a reduction ofAβ level and, therefore, BACE1 is regarded as an attractiveand plausible target in AD prevention.

Allomyrina dichotoma is a precious insect found ineastern Asia (China, Japan and Korea). The larval form ofA. dichotoma has been used as a traditional medicine forvarious liver diseases and diabetes mellitus (Taketa et al.1986; Lee & Lee 2009). Many studies have demonstrated

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that A. dichotoma possesses diverse biological functionsincluding antineoplastic, antibacterial and antioxidanteffects (Sagisaka et al. 2001; Kim et al. 2007; Suh et al.2010). Despite its various biological effects, the anti-dementia property of A. dichotoma has not been studied sofar. Therefore, the present study was designed to demon-strate the anti-AD effect of A. dichotoma larvae through theregulation of in vitro BACE1 and Aβ(25–35)-induced tox-icity in rat adrenal pheochromocytoma PC12 cells.

Materials and methods

General

Aβ(25–35) and 3-[4,5-dimethyl-thia-zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained fromSigma Chemical Co. (St. Louis, MO, USA). Roswell ParkMemorial Institute (RPMI) 1640 medium and phosphate-buffered saline (PBS) were purchased from HycloneLaboratories, Inc. (Logan, UT, USA). N2 supplement, fetalbovine serum, and RPMI 1640 phenol red free medium werepurchased from Gibco BRL (Grand Island, NY, USA).5-[and-6]-Chloromethyl-2′,7′-dichlorodihydrofluoresceindiacetate, acetyl ester (CM-H2DCFDA) and Hoechst 33342dye were obtained from Molecular Probes Inc. (Eugene, OR,USA). All other chemicals used were of the highest gradecommercially available. Resveratrol (99% pure), a positivecontrol, was purchased from Sigma-Aldrich (St. Louis, MO,USA).

Extraction and fractionation of

A. dichotoma larvae

The freeze-dried powder of A. dichotoma larvae was pro-vided by World Way Corp. (Chungnam, Korea). The powder(0.5 kg) was refluxed with 95% ethanol (EtOH; 10 L, roomtemperature) twice and the concentrated EtOH extract(83.80 g) was consecutively partitioned with hexane, ethylacetate (EtOAc) and H2O to give 56.01 g, 0.61 g and22.41 g, respectively.

Enzyme assays

BACE1 and TACE assays were carried out according to thepreviously reported methods with slight modifications(Youn & Jun 2012). Other serine proteases, chymotrypsinand trypsin, were assayed according to our previous methodsusing N-benzoyl-L-Arg-pNA and N-benzoyl-L-Tyr-pNA assubstrates, respectively (Youn & Jun 2012).

Cell culture

PC12 cells were grown in RPMI 1640 medium sup-plemented with 10% heat-inactivated horse serum, 5%

fetal bovine serum at 37°C in a humidified 95% air/5% CO2

incubator.

Determination of cell viability

Aβ(25–35) was dissolved in dimethyl sulfoxide (DMSO)and stored at −20°C. For aggregation, the stock solution wasdiluted with PBS and incubated at 37°C for 48 h prior toeach assay. The final concentration of DMSO in sampleswas less than 0.01%, which did not affect cellular viability(data not shown). The method of MTT assay by Hong et al.(2012) was adopted with some modification. Cells wereplated at the density of 1 × 104 cells/well. After incubationwith the sample for 1 h, the cells were treated with 50 μMAβ(25–35) for 24 h. Thereafter, 0.5 mg/mL of MTT reagent(final concentration) was added, incubated and then theformazan reduction product was determined using amicroplate reader at a wavelength of 570 nm (ELX808,Biotek, Winooski, VT, USA).

Determination of intracellular reactive oxygen

species (ROS) accumulation

To monitor intracellular ROS generation, the fluorescentprobe CM-H2DCFDA was used. After treatment with 50 μMAβ(25–35) for 24 h in the presence or absence of sample,cells (1 × 104 cells/well on 96 well plates) were maintainedin serum-free N2 medium with 10 μM CM-H2DCFDA at37°C for 30 min. The medium was removed and Hank’sbalanced salt solution (HBSS) was added to each well. Fluo-rescence intensity was measured using an excitation wave-length of 485 nm and an emission wavelength of 528 nmusing a fluorescence spectrophotometer (FLX800, Biotek,Winooski, VT, USA).

Apoptotic morphology observation by Hoechst

33342 staining

PC12 cells (1 × 106) cultured on cover slips were plated in6-well plates and exposed to 50 μM Aβ(25–35) and samplesfor 24 h. After treatments, the cells were fixed with 4%formaldehyde in PBS and stained with 1 μg/mL Hoechst33342. Hoechst-stained cells were observed and photo-graphed under a fluorescent microscope (Olympus, Tokyo,Japan).

Determination of caspase-3 activation

The activity of caspase-3 was determined by the caspase-3assay kit according to the protocol supplied by the manufac-turer (BioVision, Mountain View, CA, USA). Cultured PC12cells (1.5 × 106) were lysed for 10 min in an ice bath, and thelysates were centrifuged at 2500g (5000 rpm) for 5 min at

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4°C. The supernatant was incubated with substrate peptidein 50 μL of incubation buffer at 37°C for 1 h. Absorbance ofthe chromophore p-nitroaniline (pNA) produced was meas-ured by reading absorbance at 405 nm in a microplatereader.

Statistical analysis

Data were expressed as mean ± SD (n = 3). Statistical dif-ferences between groups were performed by analysis ofvariance (ANOVA) with Student’s t-test and Duncan’smultiple range tests using the Statistical Analysis System(SAS). A value of P < 0.05 was considered statisticallysignificant.

Results and discussion

BACE1-specific inhibitory effect of

A. dichotoma larvae

To evaluate the potential of A. dichotoma larvae as anti-ADagents, BACE1 inhibitory activity was measured. As shownin Figure 1, the EtOH extract of A. dichotoma larvae exerted38.65 ± 1.35% of BACE1 inhibitory activity at 50 μg/mL.Upon fractionation of the A. dichotoma larvae EtOH extract,hexane, EtOAc and H2O fractions were given. Among foursolvent fractions, the EtOAc fraction exhibited the predomi-nant BACE1 suppressing effect of 52.55 ± 4.97% at50 μg/mL [half maximal inhibitory concentration (IC50)9.2 × 10−5 μg/mL].

For determination of enzyme specificity, the inhibitoryeffects of A. dichotoma larvae against TACE and otherserine proteases were compared with that of BACE1. As

shown in Table 1, none of the extract and fractions ofA. dichotoma larvaeexhibited any inhibitory effects againstTACE or serine proteases including trypsin andchymotrypsin, demonstrating that A. dichotoma larvaeEtOH extract and solvent fractions possess relatively selec-tive and specific inhibitory effects against BACE1.Resveratrol did not exhibit any significant inhibition againstthe above enzymes up to the concentration of 100 μΜ.

Peptidomimetic BACE1 inhibitors, such as amino-ethylenes, octapeptide, OM99-1, OM99-2 etc., have beensynthesized (Silvestri 2009). Despite their dramatic BACE1inhibitory effects, these synthetic BACE1 inhibitors havetrouble with stability and blood brain barrier penetration.However, BACE1 inhibitors from natural resources are

Figure 1 β-Secretase inhibitory activityof ethanol (EtOH) extract and solvent frac-tions from Allomyrina dichotoma. Sameconcentrations of each fraction or extractwith different letters are significantly dif-ferent at P < 0.05.

Table 1 Inhibitory activity† of ethanol (EtOH) extract and solventfractions against α-secretase and other serine proteases

Conc.(μg/mL) Trypsin Chymotrypsinα-Secretase

(TACE)

EtOH extract50 6.70 ± 0.36 5.73 ± 0.20 6.50 ± 0.25100 5.35 ± 0.33 4.27 ± 0.10 4.55 ± 0.66

Hexane fraction50 2.61 ± 0.65 3.57 ± 0.10 5.13 ± 1.22100 1.73 ± 0.40 3.37 ± 1.02 5.22 ± 1.09

EtOAc fraction50 1.89 ± 0.90 4.06 ± 0.59 8.01 ± 1.68100 1.90 ± 1.40 3.71 ± 1.11 7.27 ± 2.07

H2O fraction50 5.99 ± 0.87 2.03 ± 0.10 8.28 ± 0.99100 5.03 ± 0.71 1.15 ± 0.89 3.74 ± 1.57

†Not significant at P < 0.05.

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exclusively superior in terms of safety, chemical diversity,metabolic stability and synergetic effects in interacting withother components compared with synthetic ones (Suh &Checler 2002). Natural BACE1 inhibitors have been identi-fied from green tea, grape vine (Vitis vinifera), pomogranate,fuki (Petasites japonicus), Cornus fruit (Corni fructus), crabshell etc. (Jeon et al. 2003; Byun et al. 2005; Kwak et al.2005; Song et al. 2008; Choi et al. 2009; Youn & Jun 2012),most of which are plants. Here, we report A. dichotomalarvae as a new class of potential BACE1 inhibitors, whichwould stand as a starting point to encourage the use ofinsects as non-peptidyl natural BACE1 inhibitors in the pre-vention and/or alleviation of dementia.

Protective effect of A. dichotoma larvae against

Aβ(25–35)-induced cell death

Exposure of 50 μM Aβ(25–35) decreased cell viability to60.83 ± 0.53% compared with the control without Aβtreatment (P < 0.001) (Fig. 2). Reduction of cell viabilitycaused by Aβ was significantly restored by pretreatmentwith EtOH extract or fractions of A. dichotoma larvaein a concentration-dependent manner. Particularly, theEtOAc fraction at 50 μg/mL almost completely inhibitedthe cytotoxicity evoked by Aβ(25–35). Interestingly, theeffectiveness of EtOAc fraction (90.25 ± 0.51%) at50 μg/mL against Aβ was significantly better than that of50 μM resveratrol, a known natural anti-dementia agent(P < 0.001).

Protective effect of A. dichotoma larvae

against Aβ(25–35)-induced intra-cellular

ROS accumulation

Many studies have demonstrated that oxidative stressexceeding the potency of the endogenous antioxidantdefense systems is considered to be a detrimental factor inthe incidence of AD (Markesbery 1997). The brain is rela-tively weaker than other organs in terms of oxidative damagebecause it is exposed to higher oxidative stress but possessesa relatively low level of antioxidant defense system (Olanow1993; Reiter 1995).

Intracellular ROS levels were evaluated for determiningwhether A. dichotoma larvae can attenuate cell death byinhibiting ROS generation. ROS levels in PC12 cells weresignificantly increased by Aβ(25–35) [43.49 ± 0.74% vs100 ± 0.83%, control vs Aβ(25–35), P < 0.001] (Fig. 3).Treatment of cells with the EtOH extract or solvent fractionsof A. dichotoma larvae considerably suppressed ROS accu-mulation in dose-dependent mode. At 50 μg/mL, the EtOHextract, hexane, EtOAc and H2O fraction significantlyreduced Aβ(25–35)-stimulated ROS generation (70.05 ±2.21%, 64.40 ± 0.56%, 56.18 ± 0.635%, and 79.00 ± 1.92%,respectively, P < 0.001). In particular, the EtOAc fractionexerted the strongest inhibitory activity, which was signifi-cantly better than that of 50 μΜ resveratrol (P < 0.05). Noneof the extract or fractions of A. dichotoma larvae produceddirect reactions with CM-H2DCFDA to cause fluorescence(data not shown).

Figure 2 Protective effects of ethanol(EtOH) extract and solvent fractions fromAllomyrina dichotoma against amyloid-βpeptide (Aβ)(25–35)-induced cell death.Rat adrenal pheochromocytoma PC12cells were pretreated with samples for1 hr and further treated with 50 μM ofAβ(25–35) for 24 h. Cell viability wasassessed by 3-[4,5-dimethyl-thia-zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)reduction assay. ###P < 0.001 vs controlgroup. ***P < 0.001, **P < 0.01, *P< 0.05vs the group treated with Aβ(25–35) alone.$$$P < 0.001 vs 50 μM resveratrol, a posi-tive control. Data represent mean ± SD ofthree independent experiments.

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Inhibitory effect of A. dichotoma larvae against

Aβ(25–35)-induced apoptosis

The association between AD and Aβ, ROS and apoptosis hasbeen widely studied (Cotman et al. 1996; Kokoszka et al.2001). ROS generated by Aβ in mitochondria may penetrateto the cytoplasm, increase cellular oxidative stress anddamage cellular components and DNA, which ultimatelycauses cell death via apoptosis (Varadarajan et al. 2000;Kadowaki et al. 2005).

To estimate the ratio of cells undergoing apoptosis, cellscontaining fragmented DNA were visualized by Hoechst33342 staining. The control group without Aβ treatmentshowed round and blue nuclei (Fig. 4A-a). However, severaldistinguishing features of apoptosis, such as nuclear frag-mentation and apoptotic cellular membrane blebbing, weredetected in Aβ(25–35)-injured cells (Fig. 4A-b). The apop-totic morphologies were significantly alleviated by theaddition of extract or fractions of A. dichotoma larvae. TheEtOAc fraction at tested concentrations completely pre-vented apoptosis in terms of the morphological appearanceof PC12 cells compared with that of the control (Fig. 4B-h,i). Damage resulting from 50 μM Aβ(25–35), whichcaused 36.28 ± 2.71% apoptosis (7.50 ± 0.24%, control),was recovered significantly by A. dichotoma larvae extractand fractions. The anti-apoptotic effect of A. dichotomalarvae was exerted in a dose-dependent pattern. The EtOAcfraction at 50 μg/mL (7.43 ± 1.69%, P < 0.001), in particu-lar, demonstrated almost complete recovery of Aβ(25–35)-induced apoptosis.

Protective effect of A. dichotoma larvae against

Aβ(25–35)-induced caspase-3 activation

Aspartic acid-specific cysteine proteases, referred to as thecaspase family, are composed of more than 10 homologuesidentified in mammalian cells (Nicholson & Thornberry1997) and caspase-3 has been suggested to play a pivotalrole in apoptosis (Jang et al. 2004). To investigate ifcaspase-3 is linked to Aβ(25–35)-induced neuronalapoptosis, caspase-3 activity in PC12 cells was evaluated inresponse to Aβ(25–35)-exposure. Caspase-3 was activated2.04-fold with the treatment of Aβ(25–35) (P < 0.001,Fig. 5). A. dichotoma larvae extract and solvent fractionssignificantly inhibited Aβ(25–35)-induced caspase-3 activa-tion (P < 0.001). Here again, the EtOAc fraction exhibitedthe strongest modulation of caspase-3 activity to the basallevel at 50 μg/mL, better caspase-3 deactivation than that ofpositive control, resveratrol. The result demonstrated thatapoptotic death of PC12 cells induced by Aβ(25–35) wereprevented by A. dichotoma larvae fractions partly by sup-pressing caspase-3 activity.

AD is a complex chronic neurodegenerative disease asso-ciated with a multitude of dysfunctional processes. Thecentral components of neuritic plaques are composed oftoxic Aβ generated from APP by the primary and specificaction of BACE1. The molecular mechanism regardingAβ-mediated cytotoxicity has not been completely revealedbut many studies have demonstrated that oxidative stress isinvolved with the pathogenesis of AD (Miranda et al. 2000;Butterfield et al. 2001; Drake et al. 2003).

Figure 3 Inhibitory effects of ethanol(EtOH) extract and solvent fractions fromAllomyrina dichotoma against amyloid-βpeptide (Aβ)(25–35)-induced intracellularreactive oxygen species (ROS) accumula-tion. Rat adrenal pheochromocytomaPC12 cells were pretreated with samplesfor 1 h and further treated with 50 μM ofAβ(25–35) for 24 h. ROS production wasmeasured using the CM-H2DCFDA fluo-rescent dye. ###P < 0.001 vs control group.**P < 0.01 vs the group treated withAβ(25–35) alone. $P < 0.05 vs 50 μMresveratrol. Data represent mean ± SD ofthree independent experiments.

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Aβ has been reported to damage redox function ofmitochondria and consequently increases ROS production.Kadowaki et al. (2005) suggested that ROS produced inmitochondria may seep through the damaged membrane tothe cytoplasm, which accumulates cellular oxidative stressand initiates apoptosis by the activation of apoptotic signal-regulating kinase 1. Excessive ROS augments BACE1expression through transcriptional upregulation of theBACE1 gene, which furthermore induces more generationof Aβ in reverse (Tamagno et al. 2006). In addition, Dyrks

et al. (1992) proved transition metals facilitate Aβ oxidation,which accelerates the aggregation process of Aβ. Consistentwith the notion, several known antioxidants such as vitaminC, E, ergothioneine, catechins, and resveratrol exertedneuroprotective effects via suppressing Aβ-induced cellulardamage (Behl 2000; Jang & Surh 2003; Jang et al. 2004;Kim et al. 2011).

In the present study, we demonstrated the dualneuroprotective functions of A. dichotoma larvae: (i) theselective and specific inhibition against BACE1; and (ii)

Figure 4 Inhibitory effects of ethanol(EtOH) extract and solvent fractionsfrom Allomyrina dichotoma on amyloid-β peptide (Aβ)(25–35)-induced apop-tosis. (A) Morphological characteristics.(B) Apoptotic cell count. Rat adrenalpheochromocytoma PC12 cells werepretreated with sample for 1 h andfurther treated with 50 μM of Aβ(25–35)for 24 h. Morphological apoptosis wasdetermined by Heochst 33342 stainingunder fluorescence microscopy (magnifi-cation×400). (a) Control, (b) 50 μM Aβ(25–35), (c) 50 μM Aβ+50 μM resveratrol,(d) 50 μM Aβ+10 μg/mL EtOH, (e) 50 μMAβ+50 μg/mL EtOH, (f) 50 μM Aβ+10 μg/mL hexane, (g) 50 μM Aβ+50 μg/mLhexane, (h) 50 μM Aβ+10 μg/mL EA,(i) 50 μM Aβ+50 μg/mL EA, (j) 50 μMAβ+10 μg/mL H2O, (k) 50 μM Aβ+50 μg/mL H2O. ###P < 0.001 vs controlgroup. ***P < 0.01 and **P < 0.01 vsthe group treated with Aβ(25–35) alone.$$P < 0.01 vs 50 μM resveratrol. Data rep-resent mean ± SD of three independentexperiments.

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neuroprotection against Aβ(25–35)-stimulated cell deaththrough ameliorating free radical generation, inhibitingmembrane and DNA damage and attenuating apoptosis bycaspase-3 deactivation. Further and more detailed activity-guided identification of the active compounds in the EtOAcfraction of A. dichotoma larvae that produce this novelneuroprotective effect is needed and is currently underway.

Acknowledgments

This research was supported by Bio-industry TechnologyDevelopment Program (2011-311006-3), Ministry for Food,Agriculture, Forestry and Fisheries, Republic of Korea. Thisresearch was partially assisted by the Brain Busan 21Program.

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