Protective Effect and Mechanism of Fruit Extract of Aegle marmelos Against Amyloid-β Toxicity in a Transgenic Caenorhabditis elegans

Abstract

Alzheimer’s disease (AD) is the most common form of dementia found in the elderly. AD is caused by the accumulation of toxic proteins including amyloid-β (Aβ). The purpose of this study was to investigate the effect of fruit extract of Aegle marmelos against Aβ toxicity in Caenorhabditis elegans. The fruit of A. marmelos has been used in a traditional Thai herb formula in fatigue patients recovering from illnesses such as fever and diarrhea. We used a transgenic C. elegans strain CL4176, which expresses the human Aβ₄₂, to investigate the effects and the mechanisms of action of the extracts against Aβ toxicity. The extract of A. marmelos significantly delayed Aβ-induced paralysis. Aegle marmelos lost the ability to delay Aβ-induced paralysis in worms fed with daf-16 ribonucleic acid interference (RNAi) bacteria, but not in worms fed with hsf-1 and skin-1 RNAi bacteria. These results indicated that daf-16 transcription factor was required for A. marmelos-mediated delayed paralysis. Aegle marmelos enhanced the level of daf-16 gene. Taken together, these results indicated that A. marmelos reduced Aβ toxicity via the DAF-16-mediated cell signaling pathway. In addition, A. marmelos reduced toxic Aβ oligomers. Aegle marmelos also displayed antioxidative effect in in vivo as it enhanced resistance to paraquat-induced oxidative stress in wild type worms. All of the results suggested that A. marmelos can protect against Aβ-induced toxicity and can be a potential candidate for the prevention or treatment of AD.

Introduction

Aegle marmelos, commonly known as Bael belonging to family Rutaceae, is widely cultivating in India and many countries in Southeast Asia including Myanmar, Bangladesh, and Thailand. ¹ Leaves, fruits, stem, and root of this plant have long been used in traditional medicine because of its medicinal properties such as astringent, antidiarrheal, antidysenteric, demulcent, antipyretic and anti-inflammatory activities. ² Leaf extract of A. marmelos has acetylcholinesterase inhibitory activity and antioxidant properties in vitro. ³ Seed extract of A. marmelos possessed antidiabetic and hypolipidemic effects in diabetic rats. ⁴ The neuroprotective effect of A. marmelos leaf extract in scopolamine-induced cognitive impairment in mice was reported. ⁵ Balakumar et al reported the antifungal activity of A. marmelos leaf extract on dermatophytes. ⁶ In vitro study showed the cytotoxic effect of A. marmelos leaves in HEP G2 cell lines indicating its potential therapeutic for solid tumors of liver. ⁷ Recently, various pharmacological activities of A. marmelos extracts have been summarized, demonstrating antioxidant, antidiabetics, antimicrobial, hepatoprotective, cardioprotective and anticancer activities of the extracts. ⁸ A. marmelos contains numerous phytochemicals such as carotenoids, phenolics, alkaloids, pectins, tannins, coumarins, flavonoids, and terpenoids. ^{1,8 -10} Although A. marmelos has various medicinal benefits, no studies have yet examined its therapeutic effect in Alzheimer’s disease (AD).

AD is the most common form of dementia in the elderly and considered as one of the great healthcare challenges of the 21st century. ¹¹ AD is characterized by the formation of amyloid plaques and neurofibrillary tangles in the brains. ¹¹ Amyloid-β (Aβ) peptides and phosphorylated tau proteins are the key components of extracellular amyloid plaques and intracellular neurofibrillary tangles, respectively. Aβ is believed to be the trigger of the pathogenesis of AD. ¹² Aging is known to be the most important risk factor for the development of AD. As life expectancy is rising, the incidence of AD is expected to increase. According to the World Alzheimer Report 2018, 50 million people worldwide were living with dementia in 2018 and this number will be tripled to 152 million by 2050. ¹³ Thus, AD is now considered a major public health concern. Currently available AD treatments as acetylcholinesterase inhibitors (donepezil, galantamine, rivastigmine) and N-methyl-D-aspartate receptor antagonist (memantine) are not neuroprotective and only alleviate symptoms. ¹⁴ Therefore, the discovery of more disease-modifying drugs is urgently needed.

Transgenic Caenorhabditis elegans was established as a model for AD more than 20 years ago by Link et al. ¹⁵ Because of its short lifespan and amenability to genetic manipulation, C. elegans has been extensively used to study the molecular mechanism of AD and to screen for potential drugs for AD treatment. Through transgenic engineering, human Aβ_1–42 can be expressed in C. elegans either in neurons or muscle cells, causing amyloid-induced toxicity. For example, a transgenic C. elegans strain CL4176 expresses Aβ in muscle cells leading to rapid paralysis, ¹⁶ while strain CL2355 expresses Aβ in the neurons leading to a deficit in odorant preference associative learning behavior. ¹⁷ In addition, the ease of performing ribonucleic acid interference (RNAi) for any gene in the C. elegans allows for identification of protein involved in the mechanism of drug action. ¹⁸

Based on the benefit of A. marmelos in traditional medicine and its promises from various scientific studies, the objective of this study was to further investigate the effect of the fruit extract of A. marmelos against Aβ toxicity. We used a transgenic C. elegans model, which exhibits Aβ-induced pathological behaviors, to evaluate the potential effect of A. marmelos for AD treatment and to determine its molecular mechanism of action.

Results

To determine whether A. marmelos can protect against Aβ-induced toxicity, a transgenic C. elegans strain CL4176, a model of Aβ toxicity, was used. In this strain, human Aβ₄₂ transgene was transferred into the worms. Upon the expression of Aβ₄₂ transgene in the muscle cell induced by upshifting temperature from 16°C to 25°C, paralysis occurs. We found that A. marmelos at a concentration of 100 µg/mL significantly delayed Aβ-induced paralysis, while the concentration of 10 µg/mL did not significantly delay paralysis (Figure 1A and Table 1). So, the concentration of 100 µg/mL of A. marmelos was used in the subsequent experiment. Epigallocatechin gallate (EGCG) used as a positive control at a concentration of 100 µg/mL significantly delayed Aβ-induced paralysis (Figure 1B and Table 1).

Figure 1

Effect of Aegle marmelos and epigallocatechin gallate (EGCG) on Aβ-induced paralysis in Caenorhabditis elegans strain CL4176. Synchronized eggs were maintained at 16°C on the 60 × 10 mm culture plates (~35 eggs/plate) containing A. marmelos 10 µg/mL or 100 µg/mL (a) or EGCG 100 µg/mL (b). The hatched worms were grown for 36 hours at 16°C followed by upshifting the temperature to 25°C to induce the transgene expression. The paralysis was scored at hourly intervals. Data are illustrated as percentage of worms not paralyzed. The number of worms used in the experiment was approximately 100 per group. Each paralysis curve was obtained from 3 independent experiments. Error bars indicated SD.

Table 1

Effect of Aegle marmelos and EGCG on Aβ-Induced Paralysis in Caenorhabditis elegans Strain CL4176.

Treatment	PT50 a ±SD	Differences (h)	Significant
Control (OP50)	30.33 ± 0.6667
A. marmelos 10 µg/mL	31.33 ± 0.6670	1	No
A. marmelos 100 µg/mL	32.67 ± 0.3333	2.34	P = 0.0387
EGCG 100 µg/mL	32.67 ± 0.3333	2.34	P = 0.0387

EGCG, epigallocatechin gallate; SD, standard deviation.

^aPT₅₀ = time duration at which 50% worms were paralyzed.

Insulin/insulin-like growth factor (IGF)-1 signaling pathway regulates various physiological processes including longevity, proteostasis, and stress response. ^{19 -21} Modification of insulin/IGF-1 signaling reduces Aβ toxicity in a transgenic C. elegans expressing human Aβ. ²² Transcription factors DAF-16, HSF-1, and SKN-1 are crucial components of insulin/IGF-1 signaling pathway. To examine whether these transcription factors are required for the protective effect of A. marmelos against Aβ toxicity, we knocked down the expression of these genes using the RNAi method. We found that the protective effect of A. marmelos against Aβ toxicity was abolished in C. elegans fed with RNAi to downregulate daf-16 (Figure 2A), but not in C. elegans fed with RNAi to downregulate hsf-1 or skn-1 (Figure 2B and C). These results indicated that A. marmelos reduced Aβ toxicity through the DAF-16-mediated signaling pathway.

Figure 2

The effect of knocking down genes on the protective effect of Aegle marmelos. (a-c) Paralysis in Caenorhabditis elegans strain CL4176 with or without A. marmelos in daf-16, hsf-1, and skn-1 knock down by ribonucleic acid interference (RNAi). (a) Paralysis in C. elegans treated with or without A. marmelos in C. elegans fed with RNAi to downregulate daf-16. Black line, C. elegans grown on daf-16 RNAi bacteria alone; dotted red line, C. elegans grown on daf-16 RNAi bacteria and treated with A. marmelos. (b) Paralysis in C. elegans treated with or without A. marmelos in C. elegans fed with RNAi to downregulate hsf-1. Black line, worms grown on hsf-1 RNAi bacteria alone; dotted red line, worms grown on hsf-1 RNAi bacteria and treated with A. marmelos. (c) Paralysis in C. elegans treated with or without A. marmelos in C. elegans fed with RNAi to downregulate skn-1. Black line, C. elegans grown on skn-1 RNAi bacteria alone; dotted red line, C. elegans grown on skn-1 RNAi bacteria and treated with A. marmelos. The number of worms used in the experiment was approximately 100 per group. P value <0.05 is considered to be significant difference between treatment group and control group.

To investigate whether A. marmelos affected gene expression in C. elegans strain CL4176, the messenger RNA (mRNA) levels of daf-16, hsf-1, hsp-16.2, and sod-3 genes were measured using quantitative real-time polymerase chain reaction (PCR). It was reported that superoxide dismutase-3 (SOD-3) protects C. elegans from oxidative stress. The results show that A. marmelos significantly increased the expression of daf-16 gene (Figure 3). The levels of mRNA of hsf-1, hsp-16.2, and sod-3 were not changed when compared with the control group.

Figure 3

The effect of Aegle marmelos on the expression of daf-16, hsf-1, hsp-16.2, and sod-3 genes in a transgenic Caenorhabditis elegans strain CL4176. The messenger ribonucleic acid level of daf-16, hsf-1, hsp-16.2, and sod-3 was determined by quantitative real-time polymerase chain reaction and normalized to the expression of actin-1. The fold change was normalized to that observed in untreated control worms. The experiments were performed 3 times. Data are expressed as mean ± standard deviation. Statistically significant differences compared with the control were considered at *P < 0.05.

To investigate whether A. marmelos reduced Aβ toxicity by reducing the level of Aβ oligomers, the most toxic species of Aβ, we performed Western blot analysis in a transgenic C. elegans strain CL4176. We used 6E10 as a primary antibody to detect Aβ. The relative densities of Aβ oligomers of 20 kDa and 25 kDa band were quantified. The results showed that A. marmelos significantly reduced the level of Aβ oligomers when compared with the control group (Figure 4(a) and (b)). No Aβ species were detected in C. elegans strain CL802 (no Aβ strain) which was used as a control strain for CL4176.

Figure 4

Effect of Aegle. marmelos on amyloid-β (Aβ) oligomers and Aβ expression in a transgenic Caenorhabditis elegans strain CL4176. (a) Western blot of Aβ species in a transgenic C. elegans strain CL4176 treated with or without A. marmelos 100 µg/mL. C. elegans strain CL802 (no Aβ strain) was used as a control strain for CL4176. Arrows indicate Aβ oligomers (20 and 25 kDa). (b) Band intensities of 20 kDa oligomer and 25 kDa oligomer were analyzed by Image J software. data are expressed as mean intensity of indicated band from 3 experiments. (C) The messenger ribonucleic acid (mRNA) level of Aβ was quantified using quantitative real-time polymerase chain reaction in a transgenic C. elegans treated with or without A. marmelos. The mRNA level of Aβ was normalized to the expression of actin-1. Results are the average of 3 independent experiments. Error bars represent standard deviation.

To determine whether the reduction of Aβ-induced toxicity by A. marmelos resulted from the reduction of Aβ expression, we performed quantitative real-time PCR to measure the expression of Aβ transgene in C. elegans strain CL4176. We found that A. marmelos did not reduce the expression of Aβ transgene when compared with the control group (Figure 4(c)).

Because oxidative stress has been implicated in the pathogenesis of AD and can lead to neuronal death, ²³ we examined if A. marmelos could increase oxidative stress resistance in N2 wild-type C. elegans. Paraquat, which can increase the level of reactive oxygen species (ROS) in the worm to a point where survival is decreased, ²⁴ was used as a ROS-generating compound in the experiment. If A. marmelos has antioxidative effect, it should lengthen the survival of the worm. We found that A. marmelos 10 and 100 µg/mL significantly increased oxidative stress resistance when compared with control with P values equal to 0.0001 and 0.0008, respectively (Figure 5).

Figure 5

Effect of Aegle marmelos on the survival of wild-type (N2) Caenorhabditis elegans under oxidative stress induced by paraquat 100 mM. Graph demonstrates survival curves of control (OP50), A. marmelos 10 µg/mL-treated worms, and A. marmelos 100 µg/mL-treated worms after exposure to paraquat 100 mM. Statistically significant difference between A. marmelos 10 or 100 µg/mL and control group was analyzed using Log-rank (Mantel-Cox) test. The number of worms used in the experiment was 40-50 per group. Statistically significant differences compared with the control were considered at P < 0.05.

It is reported that phenolic compounds such as caffeic acid, ferulic acid, and protocatechuic acid ^25,26 exhibited prominent activity against Aβ toxicity in C. elegans. In addition to phenolic compounds, A. marmelos fruits also contain coumarins such as marmelosin (also known as imperatorin). ²⁷ Marmelosin attenuated oxidative stress in lipopolysaccharide-induced memory deficit in mouse model. ²⁸ Several studies showed that marmelosin exhibited potent acetylcholinesterase inhibitory activity, ^{29 -31} and the formation of Aβ was reported to be induced by acetylcholinesterase. ³² Therefore, high-performance liquid chromatography (HPLC) was performed to determine the components in the fruit extract.

HPLC fingerprint of the fruit extract of A. marmelos was generated to provide the chemical profile of the extract. Marmelosin was used as a reference standard. The detection wavelength was set at 254 nm to confirm the existence of marmelosin in the extract. The result revealed that the retention times of marmelosin standard and marmelosin in the extract were 12.28 ± 0.02 and 12.29 ± 0.01 minutes, respectively (Figure 6). Comparison to the retention time of the chemical standards, the phenolic compounds in the extract were hydroxycinnamic acids; namely caffeic acid, p-coumaric acid, and ferulic acid, as well as hydroxybenzoic acids, namely, gallic acid, protocatechuic acid, vanillic acid, and syringic acid. HPLC chromatograms of phenolic compounds in fruit extract of A. marmelos are illustrated in Supporting information (Supplemental Figures S1-3 and Tables S1-5).

Figure 6

High-performance liquid chromatography fingerprint of fruit extract of Aegle marmelos. The chromatogram was detected at 254 nm. Peaks of standard (STD) marmelosin (above), marmelosin in extract (middle), and co-injection of standard marmelosin and extract (below) are illustrated.

Discussion

Because of its various pharmacological effects, A. marmelos has long been used as an Ayurvedic medicine in the prevention and treatment of many diseases in India and Southeast Asia. ¹ It has long been used as a traditional medicine in Thailand in a form of combination formula with 2 other herbs, namely, Nelumbo nucifera and Jatropha multifida, to treat fatigue patients, who were recovering from illnesses such as fever and diarrhea. Aegle marmelos is composed of several bioactive compounds such as carotenoids, phenolics, alkaloids, pectins, tannin, coumarins, flavonoids, and terpenoids. ¹⁰ Caenorhabditis elegans has been widely used as a model to evaluate the effects of phytoconstituents in herbs against Aβ toxicity. In the present study, we used a transgenic C. elegans strain CL4176, which expresses human Aβ, to test the protective effect of a fruit extract of A. marmelos against Aβ toxicity and to explore the underlying mechanism of action.

A fruit extract of A. marmelos ameliorated Aβ toxicity as it significantly delayed Aβ-induced paralysis in a transgenic C. elegans. Using the RNAi method to knockdown daf-16, the gene that encodes a transcription factor DAF-16, A. marmelos failed to delay Aβ-induced paralysis, meaning that DAF-16 was required for the protective effect of A. marmelos. It is possible that A. marmelos reduced Aβ toxicity by increasing the activity of DAF-16 or increasing the expression of gene daf-16. Utilizing real-time PCR to measure the expression of daf-16, we found that A. marmelos significantly increased the expression of daf-16 gene. These results indicated that the protective effect of A. marmelos against Aβ toxicity was mediated through DAF-16 and A. marmelos increase the expression of daf-16.

DAF-16, the ortholog of human FOXO, is a forkhead transcription factors that is a key regulator of stress responses including oxidative stress and proteotoxicity in many organisms. ^21,33 DAF-16 is responsible for the upregulation of many genes whose protein products fight various forms of stress such as oxidative stress through superoxide dismutase (sod-3); thermotolerance and proteotoxicity via small heat-shock proteins (hsp-16, hsp-12.6). ^21,34 Although it was not significant, our results demonstrated that there is a trend to increase the expression of gene hsp-16.2. Nevertheless, A. marmelos did not change the expression of sod-3. Zhou et al ³⁵ also reported that Panax notoginseng decreased Aβ toxicity by upregulating skn-1 but not its downstream stress response gene hsp-16.2.

Another transcription factor that regulates gene expression in response to stress is HSF-1. ^33,36 In our study, we found that A. marmelos did not change the expression of hsf-1 gene. Moreover, A. marmelos still significantly delayed Aβ-induced paralysis after the downregulation of hsf-1 by RNAi. These results indicated that HSF-1 was not involved in the protective effect of A. marmelos against Aβ toxicity.

The C. elegans Nrf family transcription factor SKN-1 regulates genes that defend against oxidative and xenobiotic stress and genes involved in protein homeostasis. ²¹ After the downregulation of skn-1 by RNAi, A. marmelos still significantly delayed Aβ-induced paralysis, indicating that SKN-1 was not involved in the protective effect of A. marmelos against Aβ toxicity.

Increasing evidence suggests that prefibrillar soluble Aβ oligomers, rather than insoluble Aβ fibrillar aggregates found in amyloid plaque, are the most toxic species and correlated with Aβ toxicity, leading to neuronal pathology and clinical manifestation of AD. ^37,38 Therefore, we investigated whether A. marmelos decreased toxic Aβ oligomers in a transgenic C. elegans strain CL4176. Using Western blotting analysis, we found that A. marmelos significantly reduced Aβ oligomers. We then examined whether the reduction of Aβ oligomers was attributed to the effect of A. marmelos on the expression of Aβ transgene. Using real-time PCR, we found that A. marmelos did not change the expression of Aβ transgene. Therefore, A. marmelos may reduce Aβ oligomers by inhibiting oligomerization of Aβ.

Evidence indicates that insulin/IGF-1 signaling has an effect on the metabolism and clearance of Aβ. Reduction of insulin/IGF-1 signaling pathway protects model organisms from toxic Aβ aggregation. ^39,40 DAF-16 and HSF-1, downstream components of insulin/IGF-1 signaling pathway, play an important role in reducing Aβ toxicity. DAF-16 regulates the aggregation process to create less toxic high molecular mass that might subsequently be secreted from the cell, whereas HSF-1 modulates Aβ disaggregation process. Using the RNAi method to knock down these genes, our results revealed that DAF-16 was required for the protective effect of A. marmelos whereas HSF-1 was not required. So, we speculated that the reduction of Aβ oligomers may be more related to the effect of DAF-16 than those of HSF-1.

Oxidative stress has been hypothesized to play a role in the pathogenesis of AD by not only disrupting proteostasis but also affecting lipids, membranes, and deoxyribonucleic acid (DNA). ³³ It has been shown in the in vitro study that fruit pulp extract of A. marmelos has antioxidant activity. ^9,41 To test the effect of A. marmelos on oxidative stress in vivo in our study, we performed resistance assays using paraquat, a herbicide that can lead to the formation of ROS. ⁴² Paraquat causes the formation of toxic hydrogen peroxide to increase intracellular superoxide level. The results revealed that wild-type worms under the treatment of fruit extract of A. marmelos were more resistant than the control group to the oxidative stress.

The HPLC results verified that several phenolic compounds and marmelosin were constituents of A. marmelos fruit extract. Protocatechuic acid, ferulic acid, and gallic acid are phenolic compounds that have been proved to inhibit Aβ oligomerization, increase tolerance of oxidative stress, and prolong lifespan. ^25,26,43 Additionally, in vitro studies exhibited that phenolic compounds, namely, caffeic acid and p-coumaric acid, had the potential to minimize oxidative stress and inflammatory responses and significantly inhibited acetylcholinesterase activity. ^44,45 There have been several studies showing that marmelosin exhibits a potent acetylcholinesterase inhibitory activity. ^{29 -31} Biochemical studies indicated that acetylcholinesterase induces amyloid fibril formation and form highly toxic acetylcholinesterase-Aβ complexes. ⁴⁶ It is conceivable that the fruit extract of A. marmelos demonstrating the activity against Aβ toxicity in C. elegans was at least attributed to the presence of phenolic compounds and marmelosin.

In conclusion, our results demonstrated for the first time that A. marmelos protected against Aβ toxicity in the model organism C. elegans. A. marmelos reduced Aβ toxicity via daf-16 mediated cell signaling pathway. Furthermore, A. marmelos reduced Aβ oligomers and displayed antioxidative effect against paraquat-induced oxidative stress. These results provided valuable evidence for the future use of A. marmelos in the development of agents for the prevention or treatment of AD. Further studies need to be conducted in mammals to confirm its effectiveness.

Experimental

Plant Extract and Treatment

Dry fruits of A. marmelos (L.) Correa (Rutaceae) were purchased from Vejpongosot Co., Ltd, Bangkok, Thailand. Quality control of the fruits was determined according to Thai Herbal Pharmacopoeia 2019. The sample passed the loss-on-drying test, water extractive test, ethanol extractive test, and total ash test. The fingerprint using thin-layer chromatography was performed to identify the phytochemical constituents in the sample. The fruit sample was deposited to the herbarium of the Department of Pharmacognosy, Srinakharinwirot University, Thailand.

The powder (100 g) of dry fruits was placed in deionized water (2000 mL) and boiled for 5 minutes. The supernatant and the sediment were separated by vacuum filtration. The supernatant was subjected to spray drying to remove water using a Buchi mini spray dryer B-290 (Buchi labortechnik AG, Flawil, Switzerland). The conditions were as follows: inlet temperature 155°C, outlet temperature 122°C, feed rate of 5 mL/min, airflow rate of 30 m³/hour, and spray flow rate of 473 L/hour. The powder weighed 44 g after water was removed. The content of marmelosin, an active coumarin in A. marmelos measured by high-performance thin-layer chromatography ⁴⁷ was 9.85 µg/100 mg powder.

Stock solutions of fruit extract of A. marmelos and EGCG were made with distilled water. Aegle marmelos extract and EGCG were added directly to the OP50 food source to a desired final concentration. The treatment was given to the worms from the egg stage onward.

C. elegans Strain and Maintenance

The wild-type C. elegans strain N2 and the transgenic strain CL4176 and CL802 were obtained from the Caenorhabditis Genetic Center (University of Minnesota). The worms were maintained at 20°C (for N2) and 16°C (for CL4176 and CL802) on a nematode growth media (NGM) plate with Escherichia coli strain OP50 as a food source.

Paralysis Assay

Transgenic C. elegans strains CL4176 and CL802 were synchronized onto the 60 × 10 mm culture plates spotted with OP50 containing either a vehicle or plant extracts. Aβ transgene expression in muscle cells was induced by upshifting the temperature from 16°C to 25°C at the 36th hour after egg laying and maintained at 25°C until the end of the paralysis assay. After temperature upshift for 24 hours, the number of paralyzed worms was scored under the microscope at hourly intervals until the last worm became paralyzed. To identify the paralysis, each worm was gently touched with a platinum loop. The worm was considered paralyzed if it did not move or moved head only after touching.

RNA Interference

RNAi was performed in C. elegans by feeding the worms with double-stranded RNA(dsRNA)-containing bacteria. Caenorhabditis elegans was fed with E. coli strain HT115 expressing dsRNA specific to daf-16 or hsf-1 or skn-1 gene (Source BioScience, United Kingdom). Young adult worms were placed on NGM plate containing ampicillin (100 µg/mL), isopropyl β-d-1-thiogalactopyranoside (1 mM), and dsRNA-containing bacteria. After 3-4 hours, worms were removed and eggs were permitted to mature to L4 young larvae. These worms were considered as the first generation (F1). The L4 larvae (F1) were transferred to another plate containing dsRNA and allowed to lay eggs. The resultant adult worms were considered as the second generation (F2) and were used for the paralysis assay.

RNA Extraction and Real-Time PCR

Transgenic C. elegans strain CL4176 worms were incubated at 16°C until they reached the L3 stage, then upshifted to 25°C, and harvested at 40 hours after being upshifted. Worms were washed by using phosphate-buffered saline (PBS) and collected. Worm pellets were snap frozen in liquid nitrogen and thawed 2 times. RNA extraction was performed by following the Trizol/RNeasy hybrid RNA extraction protocol using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized using qScript XLT cDNA SuperMix (Quanta Biosciences) according to the manufacturer’s instructions. Real-time PCR was performed in a PCRmax ECO 48 Real-time qPCR system (United Kingdom) using PerfeCTa SYBR Green FastMix (Quanta Biosciences). The primers used in the quantitative PCR are listed in Table 2. Cycling conditions were 95°C × 3 minutes, followed by 40 cycles of 95°C × 5 seconds + 55°C ×15 seconds. The relative quantification of mRNA was analyzed using the 2^-rrCt method. ⁴⁸

Table 2

Primers Used in the Quantitative Real-Time Polymerase Chain Reaction.

Gene	Primer name	Primer sequence
act-1	act-1 (F)	5′-CCAGGAATTGCTGATCGTATGCAGAA-3′
	act-1 (R)	5′-TGGAGAGGGAAGCGAGGATAGA-3′
Aβ transgene	Aβ transgene (F)	5′-CCGACATGACTCAGGATATGAAGT-3′
	Aβ transgene (R)	5′-CACCATGAGTCCAATGATTGCA-3′
daf-16	daf-16 (F)	5′-AAAACTGCAGAGTACAGCAATTCCCAAATGAAA-3′
	daf-16 (R)	5′-CCCAAGCTTAATTGGATTTCGAAGAAGTGGAT-3′
hsf-1	hsf-1 (F)	5′-AAAACTGCAGGAAAAAAAGTAGGAGCAAAAAAT-3′
	hsf-1 (R)	5′-CGGGGTACCAGTCAAAAAGCTGAAAAAATCGG-3′
hsp 16.2	hsp16.2 (F)	5′-CGGGGTACCATTCAGCAGATTTCTCTTCGACGATT-3′
	hsp 16.2 (R)	5′-CCGCTCGAGTGTCACTTTACCACTATTTCCGTCC-3′
sod-3	sod-3 (F)	5′-CGGGGTACCAGCTCCTTTTAAATTAAGACA-3′
	sod-3 (R)	5′-CCGGTCGAGTATTCTTCCAGTTGGCAAT-3′

Western Blot Analysis

Aβ species in the transgenic C. elegans strain CL4176 were identified by immunoblotting using a Tris-Tricine gel. Caenorhabditis elegans strain CL802 was used as a negative control of the expression of Aβ. After treatments, the worms were collected by washing 3 times with 1× PBS and snap frozen in liquid nitrogen. The worms were sonicated in lysis buffer (20 mM Tris–hydrochloric acid [HCL] pH 7.5, 150 mM sodium chloride [NaCl], 1 mM ethylenediaminetetraacetic acid, 1 mM egtazic acid, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 µg/mL leupeptin) with 1× protease inhibitor cocktail. Protein concentration was measured using Bradford assay. Equal amounts of total protein (15 µg) of each sample were mixed with loading buffer containing 5% mercaptoethanol. Samples were heated at 95°C for 5 minutes and loaded on the gels. Gels were transferred to polyvinylidene difluoride membranes. Membranes were blocked with a solution of 5% milk in tris-buffered saline tween-20 (100 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. The membranes were then probed with a primary anti-β-amyloid antibody 6E10 (1:1000; Biolegend) or α-tubulin (1:1000; Sigma) overnight at 4°C. Antimouse immunoglobulin-G peroxidase conjugate (1:2000, Sigma) was used as a secondary antibody. Blots were visualized with an ImageQuant LAS 4000 mini (GE Health), and mean densities of bands were analyzed using ImageJ software.

Paraquat Assay (Oxidative Stress Resistance Assay)

Oxidative stress resistance assay was performed following the method by Possik et al. ⁴⁹ Caenorhabditis elegans strain N2 (wild type) was treated with vehicle (control) or A. marmelos 10 or 100 µg/mL at 20°C. Synchronized larvae were cultured at 20°C until they reached the L4 stage. Worms were transferred to 40 µL of paraquat 100 mM (0.025 g of paraquat dissolved in 1 mL of M9 buffer) in each well of a 96-well plate. Worms (5-8) were transferred to each well. The viability of the worms was scored hourly. The worms were counted as dead when they failed to respond to the gentle touch with a platinum loop.

HPLC Analysis

Reference standard marmelosin (Sigma-Aldrich, St. Louis, MO, USA) was accurately weighed and dissolved in HPLC grade methanol to get a 100 µg/mL final concentration. A high-performance liquid chromatograph (Model LC-2030C-3D, Shimadzu, Japan) coupled with a photodiode array detector was used for the analysis of marmelosin in the standard solution and the extract. The stationary phase consisted of reverse phase HiQ sil C18W column (4.6 mm × 250 mm, 5 µm) (Tokyo, Japan) and the mobile phase consisted of methanol:water (66:34, v/v) with a flow rate of 1.0 mL/minute. A 20 µL volume of standard solutions of marmelosin (100 µg/mL) was injected through the injection port of HPLC in triplicate. The detection wavelength was set at 254 nm.

Identification of Phenolics and Flavonoids in the Extract

The component of phenolics and flavonoids in the fruit extract of A. marmelos was determined by using HPLC. Phenolics and flavonoids in the extract were identified by comparing the retention time to standards. The phenolic standards used in the experiment were gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid. Rutin was used as a flavonoid standard. An HPLC (Model LC-2030C-3D, Shimadzu, Japan) coupled with a diode array detector was used for the analysis of phenolics and flavonoids in the extract according to the method by Tamprasit et al. ⁵⁰ The detector wavelength was set at 280, 320, and 370 nm. Components in the extract were identified by comparing their retention times and spectral patterns with those of standard compounds and were confirmed by using a co-injection of the standard with the extract.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5. Significant differences were assessed by one-way analysis of variance(ANOVA), followed by Dunnett’s test. Paralysis and survival curves were plotted and analyzed using GraphPad Prism 5. Value of P < 0.05 was considered statistically significant.

Supplemental Material

Supplementary Material 1 - Supplemental material for Protective Effect and Mechanism of Fruit Extract of Aegle marmelos Against Amyloid-β Toxicity in a Transgenic Caenorhabditis elegans

Supplemental material, Supplementary Material 1, for Protective Effect and Mechanism of Fruit Extract of Aegle marmelos Against Amyloid-β Toxicity in a Transgenic Caenorhabditis elegans by Roongpetch Keowkase, Nattanon Kijmankongkul, Wanapong Sangtian, Sireethorn Poomborplab, Chatpiti Santa-ardharnpreecha, Natthida Weerapreeyakul and Worapan Sitthithaworn in Natural Product Communications