Antioxidant Activity, Delayed Aging, and Reduced Amyloid-Î² Toxicity

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Antioxidant activity, delayed aging, and reduced amyloid-# toxicity of methanol extracts of tea seed pomace from Camellia tenuifolia Chia-Cheng Wei, Chan-Wei Yu, Pei-Ling Yen, Huan-You Lin, Shang-Tzen Chang, Fu-Lan Hsu, and Vivian Hsiu-Chuan Liao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503192x • Publication Date (Web): 08 Oct 2014 Downloaded from http://pubs.acs.org on October 12, 2014

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Journal of Agricultural and Food Chemistry

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Manuscript submitted to: Journal of Agricultural and Food Chemistry

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Manuscript ID: jf-2014-03192x-R1

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Antioxidant activity, delayed aging, and reduced amyloid-β toxicity of

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methanol extracts of tea seed pomace from Camellia tenuifolia

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Chia-Cheng Wei a, §, Chan-Wei Yu a, §, Pei-Ling Yen b, Huan-You Lin b, Shang-Tzen Chang b,

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Fu-Lan Hsu c, *, Vivian Hsiu-Chuan Liao a, *

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a

Sec. 4, Roosevelt Rd., Taipei 106, Taiwan

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b

Department of Forestry and Resource Conservation, National Taiwan University, No. 1 Roosevelt Road, Sec. 4, Taipei 106, Taiwan

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Department of Bioenvironmental Systems Engineering, National Taiwan University, No. 1,

c

Forest Chemistry Division, Taiwan Forestry Research Institute, 53 Nanhai Rd., Taipei 100, Taiwan

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§

Both authors contributed equally to this work.

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*

Correspondence: Vivian Hsiu-Chuan Liao, Tel: +886-2-33665239; Fax: +886-2-33663462;

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E-mail: [email protected]. Fu-Lan Hsu, Tel: +886-2-23039978 ext.2173; Fax:

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+886-2-23077306; Email: [email protected].

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1

ACS Paragon Plus Environment


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Abstract

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There is a growing interest in the exploitation of the residues generated by plants. In

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this study, we explored the potential beneficial health effects from the main biowaste, tea seed

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pomace, produced when processing tea seed. DPPH radical-scavenging and total phenolic

24

content assays were performed to evaluate the in vitro activities of the extracts.

25

Caenorhabditis elegans was used as in vivo model to evaluate the beneficial health effects,

26

including antioxidant activity, delayed aging, and reduced amyloid-β toxicity. Among all

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soluble fractions obtained from the extracts of tea seed pomace from Camellia tenuifolia, the

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methanol (MeOH) soluble fraction has the best in vivo antioxidant activities. The MeOH

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soluble extraction was further divided into 6 fractions by chromatography with a Diaion

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HP-20 column eluted with water/MeOH and fraction #3 showed the best in vitro and in vivo

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antioxidant activities. Further analysis in C. elegans showed that the MeOH extract (fraction

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#3) of tea seed pomace significantly decreased intracellular reactive oxygen species,

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prolonged C. elegans lifespan, and reduced amyloid-β (Aß) toxicity in transgenic C. elegans

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expressing human Aß. Moreover, bioactivity-guided fractionation yielded two potent

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constituents

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3-O-(2’’-glucosyl-rutinoside) and kaempferol 3-O-(2’’-xylopyranosyl-rutinoside) and both

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compounds exhibited excellent in vivo antioxidant activity. Taken together, MeOH extracts of

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tea seed pomace from C. tenuifolia have multiple beneficial health effects, suggesting that

from

the

fraction

#3

of

the

MeOH

extract,

2


namely

kaempferol

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biowaste might be valuable to be explored for further development as nutraceutical products.

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Furthermore, the reuse of agricultural byproduct tea seed pomace also fulfills the

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environmental perspective.

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Keywords: biowaste, tea seed pomace, Camellia tenuifolia, Caenorhabditis elegans, antioxidant, lifespan, amyloid-β

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Introduction

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Plant-derived products have potential pharmaceutical and nutraceutical values, yet these

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potentials are still largely unexplored. Nowadays, there is a growing interest in the

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exploitation of the plant-derived products. From an environmental perspective, it is vital to

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implement low-waste technology in the agribusiness by reusing the plant byproducts

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produced by the agro-food industry.1 However, the integral exploitation of plant-derived

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products has not yet been achieved. A number of studies have reported antioxidant activity

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from various agricultural byproducts.2,3

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The Camellia plants are cultivated as economic or ornamental plants in East Asia. The

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seeds of the Camellia plants are extracted for commercial tea oil. Some studies reported that

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the tea seed oil of Camellia oleifera has antioxidant activity and hepatoprotective effect

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against CCl4-induced oxidative damage in rats.4,5 In Taiwan, C. oleifera and C. tenuifolia are

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the two main Camellia plants. After the oil was extracted from the tea seed, the remaining tea

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seed pomace, is discarded or used as detergent or organic fertilizer with low economic value.

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A few studies reported that the tea seed pomace from C. oleifera contains large

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amounts of active compounds such as theasaponins, flavonoids, and saponin with in vitro

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biological activity.6,7 In vivo study regarding the bioactivities of the chemical compounds

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from C. oleifera tea seed pomace is limited. In contrast, the potential biological properties for

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the extraction of C. tenuifolia tea seed pomace remain to be further exploited.

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Accumulating evidences have suggested the link of aging and amyloid-β (Aß) toxicity

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with oxidative stress and mitochondrial dysfunction.8,9 Natural products have been suggested

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as promising drug candidates for Alzheimer’s Disease (AD) treatment due to their antioxidant

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properties mainly by scavenging free radical species.10 Studies on model organisms in regards

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to the effects of antioxidants (eg., vitamins, lipoic acid, coenzyme Q, resveratrol, curcumin,

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etc) supplementation on aging and longevity have been extensively reviewed.11 The model

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organism Caenorhabditis elegans has been increasingly used to study stress resistance,

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longevity, and Aß accumulation.12,13 In addition, the genetic networks of C. elegans are

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phylogenetically conserved from nematodes to vertebrates and 60%~80% of human gene

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homologues have been identified in C. elegans.14,15 This allows findings from C. elegans to be

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extrapolated and further confirmed in human.

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Given being an inexpensive and residual resource, we explored the potential beneficial

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health effects from the main biowaste, tea seed pomace, produced when processing tea seed

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from C. tenuifolia. DPPH radical-scavenging and total phenolic content assays were

79

performed to evaluate the in vitro activities of the extracts. C. elegans was used as in vivo

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model to evaluate the beneficial health effects, including antioxidant activity, delayed aging,

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and reduced amyloid-β toxicity.

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Materials and Methods

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Plant materials

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All chemicals unless otherwise stated were purchased from Sigma-Aldrich (Poole,

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Dorset, UK). Tea seed pomace of C. tenuifolia was collected in October 2011 from the tea

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tree farm located in Taichung County, Taiwan. The species were identified by the Taiwan

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Forestry Research Institute.

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Tea seed pomace extracts preparation and identification of compounds

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The dried samples (in triplicate 100-g batches) were incubated with water, hot water

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(100 oC), ethyl acetate (EtOAc), ethanol (EtOH), n-hexane (Hex), and methanol (MeOH) at

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room temperature for 1 hr (sample : solvent = 1:5). The extracts were decanted, filtered under

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vacuum, concentrated in a rotary evaporator and then lyophilized. The MeOH soluble fraction

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was further divided into 6 fractions (#1 ~ #6) by chromatography with Diaion HP-20 column

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eluted with water/MeOH (gradient elution was performed by changing from 100/0 to 0/100).

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The phytochemicals from the fraction #3 were separated and purified by

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semipreparative high-performance liquid chromatography (HPLC) on a Agilent model 1100

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pump equipped with a UV detector (Agilent 1100) and a 250 mm × 10.0 mm i.d., 5 µm Luna

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RP-21 column (Phenomenex, CA, USA). The mobile phase used was solvent A (ultrapure

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water) and solvent B (MeOH). Elution conditions were 0−5 min of 55% B (isocratic gradient);

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5−20 min of 55−65% B (linear gradient); 20−30 min of 65−100% A to B (linear gradient).

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ESI-MS data were collected using a Finnigan MAT-95S mass spectrometer, and NMR spectra

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were recorded by a Bruker Avance 500 MHz FT-NMR spectrometer. The structures of

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chemical compounds (as shown in Fig. 1) were identified by ESI-MS and NMR, and the data

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were consistent with those reported in the studies.16,17

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DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay

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The DPPH free radical scavenging activities of MeOH extracts and their derived

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fractions were measured. Extracts with a series of concentrations were dissolved in EtOH (50

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µl) and mixed with 1000 µl of 0.1 mM DPPH–EtOH solution and 450 µl of 50 mM Tris–HCl

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buffer (pH 7.4). EtOH (50 µl) was used as the control for this experiment. After 30 min of

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incubation at room temperature, the reduction of the DPPH free radical was determined by

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reading the absorbance at 517 nm. Ascorbic acid, a well-known antioxidant, was used as the

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positive control and the inhibition ratio (%) was calculated as follow:

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% Inhibition = [(absorbance of control - absorbance of test sample) / absorbance of control] x

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100. Each test was repeated three times, and the results were averaged.

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Determination of total phenolic contents

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Total phenolic contents were analyzed according to the Folin–Ciocalteu method, using

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gallic acid as a standard. The samples were dissolved in 10 ml of EtOH /water (50/50, v/v).

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Subsequently, the sample solution (0.5 ml) was mixed with 0.5 ml of 1N Folin–Ciocalteu

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reagent and then incubated at room temperature for 5 min followed by the addition of 1 ml of

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20% Na2CO3. After 10 min, the mixture was centrifuged for 8 min (12,000 g), and the

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absorbance of the supernatant was measured at 730 nm. The total phenolic contents were

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presented as gallic acid equivalents (GAE) in milligrams per gram of test sample. Each test

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was repeated three times, and the results were averaged.

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C. elegans strains and culture conditions

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C. elegans strains used in this study were Bristol N2 (wild-type); CL4176 (smg-1ts

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[myo-3/Aβ1–42 long 3′-UTR]). C. elegans was maintained and assayed (unless otherwise

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stated) at 20 oC on nematode growth medium (NGM) agar plates carrying a lawn of E. coli

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OP50. All chemicals in NGM plates and liquid are expressed as the final concentrations.

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C. elegans oxidative stress resistance assays

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Oxidative stress assays were performed as previously described.18-20 Briefly,

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synchronized wild-type L1 larvae were incubated in liquid S-basal containing E. coli OP50

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bacteria at 109 cells/ml in the presence or absence of tea seed pomace extracts (1 and 10

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µg/ml) for 72 h at 20 oC followed by 250 µM juglone (5-hydroxy-1, 4- naphthoquinone)

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(Sigma, St. Louis, MO) exposure for 3 h and then scored for viability. The survival of worms

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was determined by touch-provoked movement as described.21 Nematodes were scored as dead

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when they failed to respond to repeated touching with a platinum wire pick. The test was

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performed at least 3 independent biological replicates.

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C. elegans intracellular reactive oxygen species measurement

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C. elegans was pretreated with tea seed pomace extracts (1 and 10 µg/ml) as the

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oxidative stress assays. After 72 h at 20 oC, nematodes were washed with K-medium 3 times

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and then transferred to 500 µL of K-medium containing 100 µM CM-H2DCFDA (Molecular

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Probes, Eugene, Oregon, USA) for 2.5 h at 20 °C. At least 20 randomly selected worms from

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each set of experiments were mounted onto microscope slides coated with 3% agarose,

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anaesthetized with 2% sodium azide, and capped with cover slips. Epifluorescence images

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were captured with an epifluorescence microscope (Leica, Wetzlar, Germany) using a suited

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filter set (excitation, 480 ± 20 nm; emission, 510 ± 20 nm) with a cooled charge coupled

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device (CCD) camera. Total fluorescence for each whole worm was quantified by Image-Pro

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Plus software (Media Cybernetics, Bethesda, MD, USA).

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C. elegans lifespan assays

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Lifespan analyses were performed in the same manner for all treatments at 20 oC.

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Synchronized eggs were placed on fresh NGM plates containing UV-inactivated E. coli OP50

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in the absence or presence of tea seed pomace extracts (1 and 10 µg/ml), and nematodes were

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allowed to develop to adulthood. UV-inactivation was used to avoid the effects of live E. coli

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on the conditions of tea seed pomace extracts examined in this study.22 Surviving and dead

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worms were scored daily (starting on the first day of adulthood) until all nematodes had died.

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Nematodes that did not move when gently prodded (with a platinum wire) were judged to be

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dead. Nematodes suffering from internal hatch (a defect in egg laying) and those that crawled

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off the NGM plate were not included in the lifespan scores. During the reproductive period,

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adult nematodes were transferred to fresh NGM plates every day and then every other day

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thereafter. The test was performed at least 3 independent biological replicates.

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C. elegans paralysis assays The strain CL4176

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maintained at 16 °C was egg-synchronized onto the NGM plates

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containing E. coli OP50 in the absence or presence of tea seed pomace extracts. At the 50th hr

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after eggs laying, Aβ transgene expression was induced by upshifting the temperature from 16

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to 23°C, and continued to the end of the paralysis assays.23 Paralysis was scored at 2 h

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intervals until the last worm became paralyzed.24 The definition of paralysis of worms in this

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study was that worms exhibited pharyngeal pumping, but failed to complete one sinusoidal

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turn within 5 s of being touched on the head and tail with a platinum wire.24 The test was

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performed at least 3 independent biological replicates.

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Statistical analysis

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Statistical analysis was performed using SPSS Statistics 17.0 Software (SPSS, Inc.,

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Chicago, IL, 2008). The results are presented as the mean ± standard errors of mean (SEM).

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Comparisons and p value calculations were made between treated and untreated animals of

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the same strain using one-way ANOVA and LSD post hoc test. Differences were considered

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significant at p < 0.05 (see figures). For the lifespan and paralysis assays, animal survival was

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plotted using Kaplan–Meier survival curves and analyzed by log-rank test using GraphPad

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Prism (GraphPad Software, Inc., La Jolla, CA, USA).

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Results and Discussion

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Solvent selection

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Direct extraction using various types of solvents is the most common technique

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employed to obtain extracts with high antioxidant activity from plants. To examine the

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extraction process of compounds with antioxidant properties from tea seed pomace of C.

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tenuifolia, tea seed pomace was initially extracted by various types of solvent, including H2O

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(room temperature), hot H2O (100 oC), EtOAc, EtOH, Hex, and MeOH.

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C. elegans was used to investigate whether extracts from different solvents exert

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oxidative stress resistance. Wild-type N2 C. elegans was pretreated with different

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solvent-extracts followed by exposure to juglone-induced oxidative stress. Figure 2 showed

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that pretreatments with extracts from H2O, hot H2O (100 oC), EtOH, and MeOH significantly

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increased the survival of C. elegans exposed to juglone-induced oxidative stress (Fig. 2). It is

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noted that the extracts from hot H2O (100 oC) showed protective effect on lower dose (1

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µg/ml), whereas the higher one (10 µg/ml) did not. It is possible that the higher concentration

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of extracts from hot H2O might be toxic to alleviate the antioxidant effects on wild-type C.

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elegans. Therefore, it is likely that a linear dose-response-curve was not observed. Moreover,

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MeOH-extract exhibited the highest C. elegans survival against juglone-induced oxidative

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stress (Fig. 2). Herein, we selected extract from MeOH-extracted tea seed pomace for further

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investigation.

208 209

Fraction selection from the methanol extract

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The MeOH-extract was further fractionated by column chromatography with a Diaion

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HP-20 column, and six fractions (#1 ~ #6) were obtained. For in vitro assay, DPPH radical

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scavenging activity of MeOH soluble extract and their derived fractions of tea seed pomace

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were performed. DPPH contains a proton free radical, which significantly reduces on

214

exposure to proton radical scavengers.25 Therefore, DPPH assay has been commonly used to

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examine in vitro antioxidant activity.26 Figure 3A showed that the lowest IC50 value (the

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concentration required to inhibit radical formation by 50%) was observed in fraction #3 (IC50

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= 49.5 µg/ml), while fraction #4 (IC50 = 93.0 µg/ml) and MeOH soluble extract (IC50 = 133.4

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µg/ml) also exhibited good inhibitory effects.

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The total phenolic contents were also examined as most plant phenolics are highly

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effective free radical scavengers and antioxidants. The total phenolics in MeOH extract and

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their derived fractions were determined according to the Folin–Ciocalteu method and

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expressed as GAE. As shown in Fig. 3B, the total phenolic contents in fraction #3 showed the

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highest total phenolic contents (101.2 mg of GAE/g), followed by fraction #4 (99.9 mg of

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GAE/g) and MeOH extract (50.1 mg of GAE/g). These results indicated that the free radical

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scavenging activity of MeOH extract of tea seed pomace could be effectively enriched in

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fractions #3 and #4.

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For in vivo assay, the antioxidant activity of MeOH soluble extract and their derived

228

fractions was examined in C. elegans. Wild-type N2 C. elegans was pretreated with 1 and 10

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µg/ml of MeOH soluble extract and their derived fractions. Figure 3C showed that

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pretreatment with fractions #1 and #3 significantly enhanced the survival of worms. Although

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fraction #4 pretreatment did not show statistically increased survival of nematodes (p =

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0.0716 for 1 µg/ml; p = 0.053 for 10 µg/ml), the observed protective effect to C. elegans

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against juglone-induced oxidative stress was obvious (Fig. 3C). In contrast, fractions #2, #5,

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and #6 did not produce a significant increase in the survival of worms exposed to

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juglone-induced oxidative stress. Furthermore, it was observed that the fraction #3 (both 1

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and 10 µg/ml) exhibited the best performance in oxidative stress resistance in C. elegans.

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Taken together, the aforesaid results suggested that the free radical scavenging effect of

238

each fraction and their phenolic contents correlate closely with their oxidative stress

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resistance in C. elegans. Moreover, the #3 fraction of MeOH extract from tea seed pomace

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not only had the highest phenolic contents but also had the best antioxidant activity in both in

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vitro and in vivo tests (Fig. 3). Accordingly, fraction #3 was selected for further investigation

242

in the C. elegans model.

243 244

Fraction #3 from the methanol extract of tea seed pomace reduces intracellular ROS

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production in C. elegans

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To gain insight in which fraction #3 from the MeOH extract suppresses the

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juglone-induced oxidative stress in C. elegans, intracellular ROS production in C. elegans

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after fraction #3 pretreatment was measured. Figure 4 showed that both 1 and 10 µg/ml of

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fraction #3 significantly inhibited the production of ROS compared to that in the untreated

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control (p < 0.001) (Fig. 4). This suggests that pretreatment of fraction #3 from the MeOH

251

extract may reduce juglone-induced toxicity as observed in Fig. 3, by decreasing the levels of

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intracellular ROS in C. elegans.

253 254

Fraction #3 from the methanol extracts of tea seed pomace significantly prolongs C.

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elegans lifespan

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Aging has been correlated to oxidative stress in C. elegans.27 An increased lifespan was

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shown to be closely associated with increased survival under oxidative stress.21,28 Therefore,

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we evaluated whether fraction #3 from the MeOH extract of tea seed pomace has effect on the

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lifespan of C. elegans or not.

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The lifespan of untreated (control) and fraction #3-treated wild-type nematodes was

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compared. Adult wild-type nematodes grown on control NGM plates and fed with UV-killed

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bacteria at 20 oC have a median lifespan of 16 days with maximum lifespan of 24 days (the

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age at death of the oldest animals). When nematodes grown on medium containing fraction #3

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(1 and 10 µg/ml) and fed with UV-killed bacteria at 20 oC, fraction #3-treated worms caused a

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statistically significant increased lifespan compared to that untreated ones (log-rank, p =

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0.0372 for 1 µg/ml; p = 0.0044 for 10 µg/ml, treated vs. untreated control) (Fig. 5). The

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median lifespans for both 1 µg/ml and 10 µg/ml of fraction #3-treated worms are 20 days,

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respectively with maximum lifespan of 27 days (1 µg/ml) and 30 days (10 µg/ml) (Fig. 5).

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Taken together, the results suggest that fraction #3 from the MeOH extract of tea seed pomace

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has a beneficial effect on C. elegans in longevity.

271 272

Fraction #3 from the methanol extracts of tea seed pomace delays amyloid-β-induced

273

paralysis in transgenic C. elegans expressing human Aß

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Production of oxidative stress by amyloid-β (Aβ) is a possible cause for

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neurodegenerative diseases such as Alzheimer's disease (AD).29,30 It has previously been

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shown that certain small molecules inhibit Aβ oligomers thereby reducing its toxicity.31,32

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However, most of these studies were conducted in vitro. In addition, it is time- and

278

cost-consuming using a transgenic mice model of AD for pharmacological evaluation and

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mechanistic studies. Therefore, C. elegans has been increasingly used as model of

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neurodegenerative diseases as it offers experimental advantages to address many basic

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cellular processes / functions that are conserved among animals.33 Ginkgo biloba extract EGb

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761 and aqueous coffee extracts were previously shown to associate Aβ toxicity with

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Aβ-induced pathological behaviors in C. elegans.24,34

284

To examine whether fraction #3 has protective effects on AD, we investigated the effects

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of fraction #3 in a C. elegans model of Aβ toxicity.23 In the transgenic C. elegans strain

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CL4176 model, temperature upshifted from 16 oC to 23 oC induces human Aβ42 expression in

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body wall muscle of C. elegans resulting in an Aβ-dependent paralysis phenotype.23 In the

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present study, synchronized eggs from transgenic CL4176 worms were placed on E. coli

289

OP50 food in the presence or absence of fraction #3 (1 and 10 µg/ml) for 50 hours at 16°C,

290

then the temperature was upshifted to 23°C to induce Aβ transgene expression. Figure 6

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showed that Aβ-induced muscle paralysis was significantly delayed in nematodes fed with

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fraction #3 (1 and 10 µg/ml) compared with the untreated controls (log-rank, p = 0.008 for 1

16


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µg/ml; p < 0.001 for 10 µg/ml, treated vs. untreated control) (Fig. 6). This suggests that

294

fraction #3 from the MeOH extract has protective effects on C. elegans against the Aβ

295

toxicity.

296 297

Constituents of fraction #3 from the methanol extracts of tea seed pomace from Camellia

298

tenuifolia

299

Constituents of fraction #3 were further determined by the bioactivity-guided

300

fractionation procedure. Semipreparative HPLC was employed to isolate phytochemicals in

301

fraction #3 from the MeOH extracts of C. tenuifolia tea seed pomace. ESI-MS and NMR were

302

applied to determine two constituents, namely kaempferol 3-O-(2’’-glucosyl-rutinoside) (23 ±

303

0.11 % of crude extracts) and kaempferol 3-O-(2’’-xylopyranosyl-rutinoside) (20.65 ± 0.25 %

304

of crude extracts).

305

In Fig. 3C, as low as 1 µg/ml fraction #3 from the MeOH extract showed good oxidative

306

stress resistance in C. elegans. Thus, we further examined the antioxidant activity of

307

kaempferol 3-O-(2’’-glucosyl-rutinoside) and kaempferol 3-O-(2’’-xylopyranosyl-rutinoside)

308

with 0.24 and 0.21 µg/ml respectively for the juglone-induced oxidative stress assays. The

309

results showed that both phytochemicals exhibited higher C. elegans survival than that of the

310

control (Fig. 7).

311

In the present study, kaempferol derivatives were identified from C. tenuifolia tea seed

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pomace. Kaempferol triglycosides have been identified from a variety of plants.35 The

313

flavonoid kaempferol is commonly found in many edible plants and used in traditional

314

medicine. Many studies have reported that kaempferol and some glycosides of kaempferol

315

have a wide range of biological activities, including antioxidant, anti-inflammatory,

316

antimicrobial, and anticancer activities.35,36 In C. elegans model, previous studies have shown

317

that kaempferol decreases the intracellular ROS level, prolongs lifespan, and attenuates

318

amyloid-ß-induced toxicity.37-39 The aforesaid studies were in good agreement with our

319

present findings (Figs. 3-6). Therefore, the extracts from C. tenuifolia tea seed pomace are

320

important and valuable to be explored for further development of potential applications.

321

In conclusion, to the best of our knowledge, this is the first report demonstrating that

322

the extracts from C. tenuifolia tea seed pomace exert multiple beneficial health effects in an

323

intact organism. The MeOH extract of tea seed pomace from C. tenuifolia significantly

324

decreases

325

amyloid-β-induced paralysis in transgenic C. elegans expressing human Aß42. Aging is

326

considered as one of the major risk factors in AD, and oxidative stress has been correlated to

327

aging and amyloid-β toxicity. Therefore, the observed delay aging and reduced amyloid-β

328

toxicity by MeOH extract of tea seed pomace might be due to its antioxidant properties.

329

Moreover, two phytochemicals, namely kaempferol 3-O-(2’’-glucosyl-rutinoside) and

330

kaempferol 3-O-(2’’-xylopyranosyl-rutinoside) were identified from the extract and exhibited

intracellular

ROS

level,

prolongs

C.

elegans

18


lifespan,

and

delays

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331

excellent in vivo antioxidant activity. Our results showed that extracts of tea seed pomace

332

from C. tenuifolia have multiple beneficial health effects, suggesting that biowaste might be

333

valuable to be explored for further development as nutraceutical products. Furthermore, the

334

reuse of agricultural byproduct tea seed pomace also fulfills the environmental perspective.

335 336 337

Notes The authors declare that no competing interests exist.

338 339 340 341

ACKNOWLEDGMENTS All nematodes strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources.

342 343

References

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Watjen, W.; Kahl, R. Effects of the flavonoids kaempferol and fisetin on thermotolerance,

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Figure Legends

452

Figure 1. Structures of two phytochemicals in fraction #3 from the methanol extracts of

453

tea seed pomace from Camellia tenuifolia. Kaempferol 3-O-(2’’-glucosyl-rutinoside) and

454

kaempferol 3-O-(2’’-xylopyranosyl-rutinoside).

455 456

Figure 2. Protective effects of tea seed pomace extracts with different solvents extraction

457

on wild-type C. elegans N2 under oxidative stress. Synchronized wild-type L1 larvae were

458

pretreated with 1 and 10 µg/ml of different solvent extracts, or 0.1 % DMSO as the solvent

459

control for 72 h at 20 oC. Nematodes were then subjected to oxidative stress assays. Tea seed

460

pomace extracts-treated (1 and 10 µg/ml) and untreated control nematodes were exposed to

461

250 µM juglone for 3 h and then scored for viability. Solvents used for the extractions were:

462

water (H2O, room temperature), hot water (H2O, 100 oC), ethyl acetate (EtOAc), ethanol

463

(EtOH), n-hexane (Hex), and methanol (MeOH). At least 3 independent biological replicates

464

were performed, and approximately 60–80 worms were scored in each experiment. Data are

465

normalized to the untreated control. Results are presented as the mean ± standard error of

466

mean (SEM). Differences compared to the untreated control were considered significant at p

467

< 0.05 (*) and p < 0.01 (**) by one-way ANOVA and LSD post hoc test.

468 469

Figure 3. In vitro and in vivo antioxidant activity of different fractions from the

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470

methanol extracts of tea seed pomace. The MeOH soluble extraction was divided into 6

471

fractions (#1 ~ #6) by chromatography with a Diaion HP-20 column eluted with water/MeOH.

472

(A) DPPH scavenging activity of each test sample was presented as percentage of DPPH

473

radicals inhibition and IC50 values (µg/ml). Each test was repeated three times, and the results

474

were averaged. (B) Total phenolic contents in each sample were expressed as gallic acid

475

equivalent (mg of GAE/g of test sample). Each test was repeated three times, and the results

476

were averaged. (C) Oxidative stress was performed as described above. At least 3 independent

477

biological replicates were performed, and approximately 60–80 worms were scored in each

478

experiment. Data are normalized to the untreated control. Results are presented as the mean ±

479

standard error of mean (SEM). Differences compared to the untreated control were considered

480

significant at p < 0.05 (*) and p < 0.01 (**) by one-way ANOVA and LSD post hoc test.

481 482

Figure 4. Effects of fraction #3 from the methanol extracts of tea seed pomace on

483

intracellular ROS production in wild-type C. elegans N2. Synchronized wild-type L1

484

larvae were pretreated with fraction #3 from the methanol extracts (1 and 10 µg/ml) of tea

485

seed pomace for 72 h at 20

486

CM-H2DCFDA for 2.5 h at 20 °C and intracellular ROS for adult animals was measured.

487

Twenty randomly selected worms from each set of experiments were directly measured the

488

total GFP fluorescence for each whole worm and quantified by Image-Pro Plus software

o

C. Subsequently, worms were treated with 100 µM

27



489

(Media Cybernetics, Bethesda, MD, USA). At least 3 independent biological replicates were

490

performed. Data are normalized to the untreated control. Results are presented as the mean ±

491

standard error of mean (SEM). Differences compared to the untreated control were considered

492

significant at p < 0.01 (**) and p < 0.001 (***) by one-way ANOVA and LSD post hoc test.

493 494

Figure 5. Effects of fraction #3 from the methanol extracts of tea seed pomace on the

495

lifespan of wild-type C. elegans N2. Synchronized wild-type L1 larvae were incubated on

496

NGM plates in the absence (control) or presence of fraction #3 (1 and 10 µg/ml), and worms

497

were allowed to develop to adulthood. Surviving and dead animals were counted daily until

498

all nematodes had died. Survival curves are of untreated control compared to fraction

499

#3-treated worms. Day 1 refers to the first day of adulthood. At least 3 independent biological

500

replicates were performed. Approximately 60 worms were scored in each experiment.

501

Statistical significance of the difference between the curves (treated vs. untreated control) was

502

demonstrated by log-rank test using the Kaplan-Meier survival analysis. Differences at the p

Antioxidant Activity, Delayed Aging, and Reduced Amyloid-Î² Toxicity

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