Neuroprotective Effects of Adlay Hull Extract Against β -amyloid-induced Neurotoxicity and Lipopolysaccharide-induced Inammatory Response

Background: Nowadays, as technology and medical treatment improvements contribute to extending the lives of human beings, it also causes many diseases like noninfectious chronic and neurodegenerative with an aging population. Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. It almost always occurs in older adults whose age is above 65 years old. AD is clinically characterized by a progressive loss of cognitive abilities. Pathologically, it is defined by the appearance of senile plaques-extracellular insoluble, congophilic protein aggregates composed of amyloid β (Aβ), resulting in oxidative stress, mitochondrial disorders, synaptic atrophy, leading to function degradation of the brain. The scientific name of adlay is Coix lacryma-jobi L., which is an annual botanical. Methods: This research uses the adlay hull, as a test sample in the experiment. The 95% ethanol extract of adlay hull (AHEE) was partitioned by ethyl acetate (AHEAE), n -butanol (AHBUE) and water (AHWE) subsequently, and the test of extracts by LPS-induce a RAW264.7 inflammatory response and Aβ 25-35 -induces dPC12 cells which cause neurotoxicity as an experimental model, respectively. Investigate the inflammatory and anti-apoptosis related protein expression. Results: The results shown that the AHEE, AHEAE and AHWE exert anti-inflammatory effects, AHWE also have anti-apoptosis effects. Through mouse macrophages inflammatory protein expression experiments, as well as inhibition of iNOS expression, resulted in inhibited nitrite production. In this study, we investigated the protective effects of AHWE against the A  25-35 -induced neurotoxicity in dPC12 cells and explored the underlying mechanism. The results showed that pretreatment with AHWE significantly attenuated cell death and the elevated Bax/Bcl-2 ratio evoked by Aβ 25-35 . Moreover, AHWE significantly inhibited Aβ 25-35 and enhanced the protein levels of Akt in dPC12 cells. These observations unambiguously suggested that the protective effect of AHWE against Aβ 25-35 -induced apoptosis in dPC12 cells was associated with the enhancement of the PI3K/Akt signaling pathway. Conclusion: The results showed that adlay hull extracts have the anti-oxidant, anti-inflammatory, anti-apoptotic and neuroprotective property. These results suggest that adlay hull extracts may have a preventive therapeutic potential in the management of


Background
Due to the rapid development of modern technology, with the advancement of medical treatment, people's average life expectancy has been extended, which has led to a rapid aging of the population. With the increase of age, chronic diseases and neurodegenerative diseases also follow. Alzheimer's disease (AD) i s a c o m m o n neurodegenerative disease that often occurs in elderly people over 65 years old, causing memory loss and a cognitive decline in patients that strongly interfere with normal daily life. The generation of oxygen free radicals, reactive oxygen species (ROS), and oxidative stress is believed to be involved in the pathogenesis of neurodegenerative disorders (Kim et al., 2011). AD is caused by the accumulation of β-amyloid (Aβ) peptide and Aβ plaques in the brain. AD is a complex disease and its neurodegenerative molecular mechanism is not yet fully understood. Growing evidence suggests a link between Aβ polymorphic nature, oligomers and fibrils, and specific mechanisms of neurodegeneration (Picone et al., 2020).
Oxidative stress, nerve inflammation, and synapse atrophy, which in turn leads to the deterioration of brain function. Aβ peptide and its aggregates are the main components of senile plaques and evidences from in vivo and in vitro experiments have shown that Aβ-induced neurotoxicity contributes to the pathogenesis and progression of AD (Citron, 2002). Accumulation of A peptide causes an increase in intracellular reactive free radicals and ROS. Increased ROS and free radicals due to the A peptide leads to accumulation of lipid peroxidation, protein oxidation, and oxidation of mitochondrial DNA (Markesbery & Carney, 1999). The underlying mechanism of Aβinduced neurotoxicity is not yet fully understood but appears to involve several p a t h w a y s a s s o c i a t e d w i t h a p o p t o s i s , w h i c h i n t u r n m a y l e a d t o t he neuronal degeneration in AD (Yao et al., 2005). AD is a heterogeneous disease with a complex pathobiology. The presence of extracellular amyloid-β deposition as neuritic plaques and intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles remain the primary neuropathologic criteria for AD diagnosis. (Long & Holtzman, 2019). Therefore, inhibition of Aβ-induced neuronal apoptosis may provide a possible approach for AD prevention and treatment.
Adlay is an annual herb of the Gramineae Coix genus, and is often used as an edible or medicinal grain in Asian countries. Adlay (Coix lachryma-jobi L.) seeds have long been used to treat warts, chapped skin, rheumatism, and neuralgia in traditional Chinese medicine (TCM). Recently, studies demonstrated its anti-inflammatory, antiproliferative, anti-tumor, and anti-allergic activities (Wang et al., 2013). Many studies have indicated that adlay has rich physiologically active ingredients, including longchain fatty alcohols, phytosterols and oleamide, which have anti-oxidation, antiinflammatory, anti-tumor, blood lipid lowering health effects (Kuo et al., 2002;Huang et al., 2009;Chung et al., 2011, Yu et al., 2011. A few studies have reported that extracts of adlay hull have neuroprotective effects. However, the effects of adlay hull extracts on neuronal cell oxidative damage and neuroinflammatoion due to the A peptide are unknown.
In this study, we investigated the preventive effect of adlay hull extracts against A peptide-induced oxidative stress by assessing free radical scavenging, cell viability, and inflammatory mediator. This study uses a cellular model to investigate the physiological changes and possible mechanisms of the adlay hull agricultural waste on neurodegenerative diseases caused by oxidative stress and inflammation. We hope this research can confirm the multi-functional nutritional and health benefits of adlay hull, and to innovate the value of agricultural products, so that agricultural waste can be reused to achieve the development of sustainable agriculture. In the future, there is an opportunity to develop agricultural waste into the elder's health food.

Experimental samples and extraction methods
The experimental samples were taken from the Taichung No. 3 Coix lacryma-jobi L. of the Caotun Adlay Production Cooperative in Nantou County. The seed coat was removed by a screening machine to obtain the adlay hull. The obtained adlay hull is made into powder with a grinder for extraction. The extraction method is that the adlay hull powder is soaked in 95% ethanol for three days as the ethanol extract (AHEE).
After suction and filtration, the ethanol is removed by a rotary evaporator. The sample is re-dissolved with deionized water and placed in a separatory funnel. Ethyl acetate and n-butanol were partitioned and extracted and then concentrated under rotary evaporator to obtain ethyl acetate partitioned extract (AHEAE), n-butanol partitioned extract (AHBUE) and water partitioned extract (AHWE). The obtained organic extracts were re-dissolved in absolute alcohol to prepare stocks, and the aqueous extract was redissolved in PBS to prepare stocks. The extracts were stored in a refrigerator at -20°C for subsequent experimental analysis.

General ingredients analysis
a. Moisture content determination Place the weight of 2 g of fresh sample (W1) into a crucible (W0) that has been weighed and dried in an oven at 105C for 24 hours to reach a constant weight (W0), and the crucible containing the sample is placed in an oven at 105C for repeated drying. Cool, weigh to constant weight (W2), and divide the weight loss by the original sample weight to obtain the sample moisture content. The moisture content (%) calculation formula is equal to [W1-(W2-W0) ] / W1×100.

b. Ash content determination
Place the weight of 2 g of the dried sample (W1) into a crucible (W0) that has been weighed and dried in a 105C oven for 24 hours to reach a constant weight, placed in a 550C ash furnace, heated for 24 hours, and then dried. Weigh the box after it has dropped to room temperature (W2). The sample ash content (%) calculation formula is equal to [ W1-(W2-W0)] / W1×100. c. Crude fat content determination Place the weight of 2 g (W) of the dried sample into a cylindrical filter paper, cover it with absorbent cotton, and then take a round-bottomed flask dried in a 105C oven for 24 hours to reach a constant weight. Take the weight (W1), and place the cylindrical filter paper into Soxhlet in the middle section of the extraction tube of the extraction device, fill the round-bottom flask with 150 mL of ether and place it in the 55C constant temperature water bath for continuous extraction for 18 hours. After extraction, remove the condenser tube and take out the cylindrical filter paper, recover the ether, put the round-bottom flask in the air extraction cabinet to volatilize the remaining ether, then put it in a 105C oven for 24 hours, and finally put it in a drying oven to reduce to the room temperature. Then weigh after cooling down to room temperature (W2). Sample lipid content (%) calculation formula is equal to [ (W2-W1) / W]×100. . They were routinely maintained in 85% RPMI 1640 medium with 2 mM Lglutamine adjusted to contain 1.5 g/L sodium bicarbonate, 10% heat-inactivated horse serum and 5% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin and differentiated with 50 ng/mL of NGF in RPMI medium with 1% FBS and penicillin/streptomycin (1% FBS-NGF medium) at 37C in a humidified atmosphere of 95% air and 5% CO2. The medium was replaced every 3 days and PC12 cells were differentiated for 7-10 days before treatments. In all experiments, dPC-12 cells were seeded into 24-well multi-plates (1×10 5 cells/well). After 24 hours, the cells were pretreated in the absence or in the presence of AHEE, AHEAE, AHBUE, AHWE (600 μg/mL) for a period of 24 hours, followed by incubating with Aβ25-35 (20 μM) for an additional 24 hours.

MTT assay
Cell viability was estimated by the MTT reduction assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. Following incubation for 24 hours at 37C and 5% CO2, the supernatant was removed and 100 μL dimethyl sulfoxide added to each well to dissolve the formazan. Absorbance was measured using the ELISA reader at a wavelength of 570 nm. Cell viability was normalized as relative percentages in comparison with untreated controls.

Nitrite assay
Trypsinize the RAW264.7 confluency cells, dilute them with culture medium to 4x10 5 cells/mL, seed them in a 96-well plate, add 100 L of cell fluid to each well, and culture in a 37C, 5% CO2 incubator for 18 to 24 hours. Remove the original medium, add the test sample and LPS (100 ng/mL) for a total volume of 100 μL/well. After incubating for 18 hours in the incubator, transfer of supernatant from each well to another 96-well plate. Add the mixed Griess reagent 100 μL/well, and react for 10 minutes in the dark.
Use an ELISA reader to detect the absorbance at 540 nm. Take the absorbance against the standard curve to calculate the nitrite content.

Superoxide Dismutase (SOD) activity determination
After collecting a certain number of cells, add 1 mL of cell lysate (20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose), centrifuge at 1500 × g for 5 minutes, and collect the supernatant at 4C for use. In a 96-well plate, add 200 L diluted radical detector and 10 L sample supernatant to each well, then add 20 L diluted xanthine oxidase to stop the reaction, react at room temperature for 30 minutes, and finally detect the absorbance at 450 nm with the ELISA reader. Use different concentrations of SOD stock as the absorbance value change of the standard to obtain the regression equation of the standard curve, and substitute the absorbance value of the sample to get the SOD activity.

Statistical analysis
Data are presented as means ± SD of three replicates. Values of P < 0.05 were considered as statistical significance by one-way analysis of variance with Duncan's multiple range test using SPSS (Statistics Package Social Science) software ® . The Student's t-test was performed to analyze differences between groups, with P < 0.01 indicating a statistically significant difference.

Component analysis of adlay hull
In this experiment, the moisture, crude fat, crude protein and ash content of dried adlay hull were measured, and then the content of carbohydrates are able to be calculated after deducting the moisture, crude fat, crude protein and ash content by 100%. Each item is expressed in percentages (%). The results are shown in Table 1. moisture 7.69%, ash 10.71%, crude fat 5.34%, crude protein 11.27%, carbohydrate 64.99%.

Yield of different extraction fractions of adlay hull
A sample of 6105 g of adlay hull, dried and ground into powder, was extracted with ethanol to obtain 12.21 g of ethanol extract with a yield of 0.2%. Then take the ethanol extract for partition extraction, and obtain 5.08 g ethyl acetate fraction extract, 1.46 g n-butanol fraction extract, and 4.21 g water fraction extract. The yields are 41.6%, 12.0%, and 34.5%, respectively which are listed in Table 2.

Antioxidant ability of different extracts of adlay hull
The results of scavenging DPPH free radicals of different extracts of adlay hull are shown in Figure 1

Antioxidant effect of different extracts of adlay hull in dPC12 cells
The SOD activity of different extracts of adlay hull in dPC12 was shown in Figure 5(a).
AHBUE has the highest SOD activity at a concentration of 600 g/mL, about 10.99 U/mL. The results of catalase activity of different extracts of adlay hull in dPC12 are shown in Figure 5(b). Different extracts of adlay hull can increase the activity of CAT.

The effect of different extracts of adlay hull with Aβ 25-35 inducing protein expression in dPC12 cells
The expression of PI3K protein of dPC12 cells after treatment with different extracts of the adlay hull increased significantly (P<0.05). In addition, AHWE activated PI3K protein and showed the best performance is shown in Figure 6(a). The expression of p-Akt protein in dPC12 cells treated with AHWE was significantly increased when compared with the control group (P<0.05). AHWE has the best effect of activating p-Akt protein is shown in Figure 6(b). The expression of Bcl-2 protein of dPC12 cells treated with AHWE was significantly increased when compared with the control group (P<0.05). AHWE has the best effect of activating Bcl-2 protein as shown in Figure 6(c).
The Bax protein expression of dPC12 cells treated with AHWE was significantly reduced when compared with the control group (P<0.05). AHWE has the best inhibitory effect on Bax protein as shown in Figure 6  Values are means ± SD (n = 3)  The values are the means ± SD (n = 3) and analyzed using one-way ANOVA, followed by Duncan's new multiple range test. Bars that do not share a common letter are significantly different (P＜0.05) from each other. Dexamethasone at the concentration of 1M was used as a positive control; cells were treated with 100 ng/mL LPS to induce inflammation.

Antioxidant ability of different extracts of adlay hull
In the in vitro antioxidant capacity test of different extracts of adlay hull, AHBUE was significantly better than other extracts in scavenging DPPH free radicals and the total phenol content. The experimental sample is the waste of adlay and the concept of waste reuse can create greater economic benefits for crops. The literature indicated that adlay contains special physiological functional components, such as coniferyl alcohol, syringic acid, ferulic acid, syringaresinol, 4-ketopinoresinol, campesterol, β-sitosterol, coixol, p-hydroxybenzaldehyde, syringaldehyde, vanillin, p-coumanc acid, gallic acid, vanillic acid, and flavonoids (naringenin, quercetin, tricin) and other phenolic compounds (Otsuka et al., 1988). Phenolic compounds are widely found in the plant kingdom. They can provide hydrogen ions in chemical reactions to inhibit the oxidation of free radicals and achieve the purpose of anti-oxidation. Flavonoids account for 80-90% of phenolic compounds. They are also widely found in the plant kingdom and have the best antioxidant capacity. DPPH-scavenging active components from adlay hulls were identified to be coniferyl alcohol, syringic acid, ferulic acid, syringaresinol, 4ketopinoresinol, and mayuenolide (Kuo et al., 2002). Our studies have found that the adlay hull contains a lot of phenolic compounds such as flavonoids, and the content of AHBUE was significantly higher than other extracts, and it also presents the best antioxidant capacity. This study also found that AHWE also has good antioxidant capacity in dPC12 cells for SOD and CAT, so it is speculated that it is caused by polyphenols.

Anti-inflammatory ability of different extracts of adlay hull
LPS induces inflammation in macrophages, and the inflammation process will acetylate the inflammatory cytokine genes (Cooper et al., 2010), it will aggravate the inflammation reaction, and a large amount of nitric oxide will be produced during the inflammation process. Nitric oxide (NO) is metabolized into nitrite (Nitrite), which acts as a mediator in the process of inflammation, enabling the synthesis of other inflammatory substances and jointly triggering the inflammatory response of the organism. In addition to their antioxidant capacity, phenolic compounds and flavonoids also inhibit LPS-induced inflammation in RAW264.7 . This study found that AHEE, AHEAE and AHWE all have the ability to inhibit the production of nitrite. Among them, AHEE and AHWE have a good ability to inhibit the production of nitrite at low concentrations, and are equal to the anti-inflammatory drug dexamethasone. NOS can be divided into three types, namely neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). It is important to note that iNOS plays an important role in the inflammation process. iNOS is mostly found in macrophages, lymphocytes and vascular smooth muscle cells, and it will synthesize a large amount of NO when stimulated by LPS (Nathan and Xie, 1994), cause cell damage and excessive vasodilation, and finally cause severe inflammation and complications (Rockey et al.,1998). The literature pointed out that the main active ingredient after purification and separation of the ethyl acetate partition extract of the adlay hull contains two currently unknown compounds and six known compounds. They are coniferyl alcohol, 4-ketopinoresinol, phenolic compounds (p-vanillin, p-hydroxybenzaldehyde) and flavonoids (tricin, 5,7-dihydroxychromone), among which coniferyl alcohol and 4ketopinoresinol can inhibit the iNOS protein expression (Choi et al., 2015). This study found that AHEE, AHEAE and AHWE have good inhibition of iNOS protein, therefore, it is speculated that the experimental samples contain active ingredients such as coniferyl alcohol and 4-ketopinoresinol, and these two known active ingredients are natural phenolic compounds.

Anti-apoptotic ability of different extracts of adlay hull
Alzheimer's disease is a serious neurodegenerative disease, the brain with amnesia is accompanied by neuronal degeneration and damage, its main pathological features are tangled nerve fibers and deposition of β-amyloid peptide (Cuenco et al., 2008). The deposition of β-amyloid peptide will form free radicals, causing initial inflammation and damage and finally nerve cell death (Su et al., 2008

Conclusions
This study found that AHBUE is significantly better than other extracts in scavenging   Effects of the different adlay hull extracts on nitrite production in RAW264.7 cells. The values are the means ± SD (n = 3) and statistical analysis was done using the Student's t-test. *P < 0.01; **P < 0.001, were signi cantly different from the control group. Dexamethasone at the concentration of 100M was used as a positive control; cells were treated with 100 ng/mL LPS to induce in ammation.

Figure 3
Effects of the different adlay hull extracts on iNOS protein expression in RAW264.7 cells. The total cellular protein (20 μg/lane) was separated on a 10% SDSPAGE, transferred to a PVDF membrane and stained with antibodies speci c for iNOS. The values are the means ± SD (n = 3) and analyzed using oneway ANOVA, followed by Duncan's new multiple range test. Bars that do not share a common letter are signi cantly different (P0.05) from each other. Dexamethasone at the concentration of 100M was used as a positive control; cells were treated with 100 ng/mL LPS to induce in ammation.    Effects of the different adlay hull extracts on ratio of Bcl-2/Bax protein expression in dPC12 cells. The values are the means ± SD (n = 3) and analyzed using one-way ANOVA, followed by Duncan's new multiple range test. Bars not sharing a common letter are signi cantly different (P0.05) from each other.
The cells were treated with 20 μM Aβ25-35 to induce neurotoxicity.