Accepted Manuscript

Antioxidant activities of aqueous extract from Stevia rebaudiana stem waste to

inhibit fish oil oxidation and identification of its phenolic compounds

Hui Yu, Gangqiang Yang, Minoru Sato, Toshiyasu Yamaguchi, Toshiki

Nakano, Yinci Xi

PII: S0308-8146(17)30569-1
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.04.004
Reference: FOCH 20874

To appear in: Food Chemistry

Received Date: 16 October 2016

Revised Date: 12 March 2017
Accepted Date: 3 April 2017

Please cite this article as: Yu, H., Yang, G., Sato, M., Yamaguchi, T., Nakano, T., Xi, Y., Antioxidant activities of
aqueous extract from Stevia rebaudiana stem waste to inhibit fish oil oxidation and identification of its phenolic
compounds, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.04.004

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Antioxidant activities of aqueous extract from Stevia rebaudiana stem waste

to inhibit fish oil oxidation and identification of its phenolic compounds

Hui Yu 1, 2,*, Gangqiang Yang3 , Minoru Sato 2, Toshiyasu Yamaguchi2 , Toshiki

Nakano2 , Yinci Xi4

1
College of Food Engineering, Ludong University, Yantai, 264025, P.R. China

2
Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555,

Japan

3
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug

Evaluation (Yantai University), Ministry of Education, Collaborative Innovation

Center of Advanced Drug Delivery System and Biotech Drugs in Universities of

Shandong, Yantai University, Yantai, 264005, P.R. China

4
College of Food Science and Technology, Shanghai Ocean University, Shanghai,

201306, P.R. China

*Corresponding Author: Hui Yu

Mailing address: College of Food Engineering, LuDong University, Yantai,

264025, P.R. China

E-mail: zoehuihui@hotmail.com

Tel: +86-535-6695491

1
Abstract

We investigated the potential for exploiting Stevia rebaudiana stem (SRS) waste

as a source of edible plant-based antioxidants finding for the first time that the hot

water extract of SRS had significantly higher antioxidant activity against fish oil

oxidation than that of the leaf, despite SRS extract having lower total phenolic

content, DPPH radical scavenging activity and ORAC values. To locate the major

antioxidant ingredients, SRS extract was fractionated using liquid chromatography.

Five phenolic compounds (primary antioxidant components in activity-containing

fractions) were identified by NMR and HR-ESI-MS: vanillic acid 4-O-β-D-

glucopyranoside (1), protocatechuic acid (2), caffeic acid (3), chlorogenic acid (4)

and cryptochlorogenic acid (5). Further analysis showed that, among compounds

2-5, protocatechuic acid had the highest capacity to inhibit peroxides formation,

but exhibited the lowest antioxidant activities in DPPH and ORAC assays. These

results indicate that SRS waste can be used as strong natural antioxidant materials

in the food industry.

Chemical compounds studied in this article:

Caffeic acid (PubChem CID: 689043); Chlorogenic acid (PubChem CID:

1794427); Cryptochlorogenic acid (PubChem CID: 9798666); Protocatechuic acid

2
(PubChem CID: 72); Vanillic acid 4-O-β-D-glucopyranoside (PubChem CID:

14132337)

Keywords: extract of Stevia rebaudiana stem, phenolic compounds, antioxidant

activity, waste exploitation, inhibition of fish oil oxidation

3
1. Introduction

Currently, the exploitation of waste materials generated by the food

processing industry (Reis et al., 2012; Roselló-Soto et al., 2015) and the use of

phenolic antioxidant-rich plant extracts as food additives and/or nutraceuticals

(Shahidi & Ambigaipalan, 2015) have both been of increasing interest. This is

because wastes of plant origin often contains natural antioxidants which are much

safer than synthetic antioxidants (Shahidi et al., 2015) and constitute a large

source of valuable compounds (Reis et al., 2012; Roselló-Soto et al., 2015).

Stevia rebaudiana (Bertoni), a perennial shrub of the Asteraceae family, is a

sweet herb native to certain regions of South America (Paraguay and Brazil), and

now consumed globally. It is also cultivated in China, Korea, Mexico, the United

States and Southeast Asia. A non-caloric natural sweetener produced from the leaf

extract has been widely used in soft drinks, soy sauce, instant noodles, yogurt and

other foods in Japan, Brazil and elsewhere (Lemus-Mondaca, Vega-Gálvez, Zura-

Bravo, & Ah-Hen, 2012). The European Food Safety Authority have recognized

the safety of the leaf extract for alimentary use since 2011. In addition, the dried

leaves also contain high levels of total phenols (Periche, Castelló, Heredia, &

Escriche, 2015), minerals and other compounds (Lemus-Mondaca et al., 2012)

with a high antioxidant capacity (Yildiz-Ozturk, Nalbantsoy, Tag, & Yesil-

Celiktas, 2015) with their potentially beneficial effects on human health. S.

4
rebaudiana leaf has been increasingly consumed as an infusions in the food and

cosmetic industry because of its high levels of antioxidant properties (Barba,

Criado, Belda-Galbis, Esteve, & Rodrigo, 2014; Gawełbęben et al., 2015). With

this wide range of applications, the production of S. rebaudiana leaf has risen to

an estimated 4000 t annually. Therefore, a large amount of waste mostly stems, is

being generated and its disposal is considered a problem so there has been an

increasing demand to exploit this stem waste materials.

Long chain polyunsaturated fatty acids (PUFA) are conditionally essential

nutrients for ensuring adequate growth, promoting health and preventing disease

in humans, one example being omega-3 PUFA (Gil, Serra-Majem, Calder, & Uauy,

2012). Fish oil, a major source of omega-3 PUFA, containing mainly all cis-

4,7,10,13,16,19-docosahexaenoic acid (DHA) and all cis-5,8,11,14,17-

eicosapentaenoic acid (EPA), is difficult to handle and store because PUFA are

vulnerable to oxidation (Zuta, Simpson, Zhao, & Leclerc, 2007). The products of

lipid oxidation have cytotoxic effects on humans so antioxidants are used to

control lipid oxidation. Because of increasing consumer concerns with health,

replacing synthetic antioxidants with those based on edible plants has attracted the

attention of the food industry. Plant extracts such as green tea, apple peel, S.

rebaudiana, rosemary and oriental herbs have been intensively studied (Sekhon-

Loodu, Warnakulasuriya, Rupasinghe, & Shahidi, 2013; Shahidi et al., 2015). Of

5
these, the hot water extract of S. rebaudiana has been shown to exhibit higher

level of antioxidant activity for inhibiting sardine oil oxidation than green tea

extract or vitamin E (VE) at the same mass concentration (Xi, Yamaguchi, Sato, &

Takeuchi, 1998a).

The present study aims to investigate the potential for exploiting S.

rebaudiana stem (SRS) waste as an edible plant-based antioxidant. We will

compare the inhibition of fish oil oxidation by the hot water extract of SRS with

that by the S. rebaudiana leaf. To locate its primary antioxidant components, the

SRS extract will be separated through an activity-guided fractionation approach

using fish oil oxidation, DPPH radical scavenging and oxygen radical absorbance

capacity (ORAC) assays. Then the chemical structures of the purified compounds

in the primary antioxidant activity-containing fractions will be identified using

nuclear magnetic resonance (NMR) and high-resolution electro spray ionization

mass spectrometry (HR-ESI-MS).

2. Materials and methods

2.1. Chemicals and reagents

6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and

Fluorescein (free acid) (FL) were supplied by Sigma-Aldrich (St. Louis, MO,

USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2´-azobis (2-amidinopropane)

6
dihydrochloride (AAPH), Folin-Ciocalteu (FC) reagent were purchased from

Wako Pure Chemical Industries (Osaka, Japan). Fish oil of Yellowfin tuna

(Thunnus albacares) was obtained from Maruha Nichiro Co. Ltd. (Miyagi, Japan).

2.2. Plant materials and preparation of extracts

S. rebaudiana stem (SRS), ground mechanically, was obtained as a fine

powder from Shandong Province, China. Aliquots were stored in polyethylene

bags at -15 °C until use.

SRS powder (500 g, dry weight) was soaked in distilled water (5 L) overnight,

boiled while stirring for 30 min and filtered. This extraction was repeated with the

residue. The filtrates were combined, concentrated under reduced pressure at

40 °C in a rotary vacuum evaporator, and lyophilized, yielding 55.8 g of dried

extract.

2.3. Separation of extracts

The stem extract (52 g) was redissolved in 260 mL of distilled water and

discolored with activated carbon (0.2%) by shaking at 60 °C for 1 h. After

filtration, the filtrate was desalted by electrodialysis (Asahi Chemical Industry

Microacilyzer Model G1, Neosepta cartridge AC-110-10), and then dialysed with

12000~14000 MW cutoff seamless cellulose tubing at 5 °C for 1 d with 12 L of

distilled water. The inner-dialysate and outer-dialysate were concentrated and

lyophilized to yield 3.2 g and 30.1 g, respectively.

7
 The outer-dialysate (30 g) was then redissolved in ultrapure water and

divided into 6 equal parts for applying to a column (500 × 20 mm i.d.) filled with

ODS (200 g, YMC, ODS-A 12 nm S-150 µm, Japan). Elution was with ultrapure

water at a flow rate of 1.5 mL min-1. Detection was with a RI-UV Monitor (YRU-

880 midget, Shimamura, Japan). Fractions A, B, C, D, E were obtained according

to absorbance at 250 nm (Fig. 2e), then separately concentrated and lyophilized to

yield 19.4 g, 1.39 g, 0.22 g, 0.34 g, 0.10 g, respectively.

2.4. Isolation and identification of phenolic compounds from fractions B and E

Fractions B and E were redissolved in ultrapure water and separated by

HPLC (PU-980, Jasco, Japan) with a preparative ODS column (TSK-GEL ODS-

80TS, 300 × 21.5 mm, 5 µm, TOSOH). Detection was with a photo diode array

detector (MD-910, Jasco, Japan) set at 250 nm. The gradient mobile phase

consisted of 2% MeCN containing 0.1% TFA (Sol. A) and 60% MeCN containing

0.1% TFA (Sol. B) at a flow rate of 10 mL min-1 . Fraction B was eluted with a

linear gradient from 0 to 100% Sol. B in Sol. A in 100 min. Fraction E was eluted

with a linear gradient from 0 to 80% Sol. B in Sol. A in 60 min. Isolated

compounds were further purified by the same semi-preparative HPLC system. The

NMR spectra of purified compounds were measured in D2 O with a JEOL JNM-

ECA400 instrument (Tokyo, Japan). HR-ESI-MS was performed with a JEOL

JMS-T100LC Accu TOF (Tokyo, Japan).

8
2.5. Determination of total phenolic contents

The total phenolic contents of SRS and leaf extracts were measured

according to the Folin-Ciocalteu colorimetric method (Singleton & Rossi, 1965),

calibrating against the gallic acid standards and expressing the results as mg gallic

acid equivalent per g of dry extract (mg of GAE g-1).

2.6. Fish oil oxidation procedure

Fish oil was oxidized using a method described previously with slight

modifications (Xi, Yamaguchi, Takeuchi, & Iida, 1995). To oxidize fish oil,

samples were prepared as follows: fish oil (1.7 g) was mixed with 0.1 g of

emulsifier (Poem PR-100) under a nitrogen atmosphere at 60 °C. Then 0.2 mL of

the SRS or leaf sample solution was added dropwise and the mixture vortexed

under a nitrogen atmosphere. The oxidation of the fish oil samples containing

antioxidants and the controls without antioxidants was induced by exposing them

to heat (50 °C) using a shaker oven operating at 120 rpm.

The primary lipid-oxidation products of fish oil were quantified using the

peroxide value (POV) assay as described by Bilinski, Jonas, and Lau (1978) with

slight modifications. Briefly, the oxidation sample (50 mg) was added to 1.8 mL

acetic acid and 1.2 mL chloroform. After adding saturated KI solution (0.5 mL),

the mixture was reacted for 1 min at RT under a nitrogen atmosphere then kept in

the dark for another 5 min. Ultrapure water (3 mL) was then added with two drops

9
of starch indicator. The titration was run against a standard solution of sodium

thiosulfate (0.01 N) with a blank also being determined under similar conditions.

The POV was calculated and expressed as milliequivalents peroxide per kilogram

of oxidation sample:

POV (meq kg-1 ) = (S - B) × 0.01F × 1000/W

where S and B are the volumes of titration (mL) in the sample and blank,

respectively, F the titre of sodium thiosulfate solution, and W the sample weight

(kg). The percentage inhibition of peroxides formation in fish oil at day 1 was

calculated using the following equation:

Percentage inhibition of peroxides formation = [1 − (POVsample/POVcontrol)] ×

100%

The secondary oxidation products of fish oil were measured using

thiobarbituric acid-reactive substances (TBARS) assay as described by Maqsood

and Benjakul (2010) with minor modifications. The oil sample (0.1 g) were mixed

with 0.9 ml ultrapure water and 5 ml of a TBA solution containing 0.375%

thiobarbituric acid, 15% trichloroacetic acid and 0.25 N HCl. The mixture was

heated in boiling water for 10 min to develop a pink colour, cooled with running

tap water and then centrifuged at 5000 g for 10 min. The absorbance of the

aqueous layer was measured at 532 nm. A standard curve was prepared using

1,1,3,3-tetramethoxypropane (malonaldehyde, MAD) at a concentration ranging

10
from 0 to 20 ppm and TBARS was expressed as mg of MAD equivalents/kg

sample. The percentage inhibition of TBARS formation in fish oil at day 1 was

calculated using the following equation:

Percentage inhibition of TBARS formation = [1 − (A sample at day 1 – A control at day

0)/(A control at day 1 – A control at day 0 )] × 100%

2.7. DPPH radical scavenging activity

The DPPH radical scavenging activity of samples was measured according to

the method described by Suda et al. (2005). Briefly, one hundred microliters of

each sample in 50% ethanol solution was pipetted into a 96-well plate, followed

by 50 µl 200 mM MES buffer (pH 6.0) and 50 µl freshly prepared 800 µM DPPH

ethanol solution. The plate was shaken for 20 min and absorbance at 520 nm was

determined using a microplate reader (Immuno Mini NJ-2300, BioTec, Japan). To

eliminate colour effects, a control was run without DPPH solution. DPPH radical

scavenging activity is expressed as trolox equivalents (TE).

2.8. ORAC assay

The ORAC assay was carried out on a Fluoroskan Ascent FL microplate

fluorometer (Thermo Fisher Scientific Oy, Vantaa, Finland). All samples were

prepared in phosphate buffer (0.075 M, pH 7.4) and analyzed according to the

following procedure (B. Ou, Hampschwoodill, & Prior, 2001). An aliquot (20 µL)

of diluted sample, trolox standard and blank (phosphate buffer) were added to a

11
96-well reading microplate followed by phosphate buffer (20 µL) and FL solution

(0.63 µmol/L, 20 µL) to each well. After preheating the microplate for 5 min at

37 °C, AAPH solution (0.128 mol/L, 140 µL) was added and measurements were

taken immediately, using an excitation wavelength of 485 nm and an emission

wavelength of 538 nm. Results are expressed as TE.

2.9. Statistical analysis

All determinations were performed in triplicate (The ORAC assay in

quadruplicate) and data are expressed as mean ± standard deviation (SD). Data

were analyzed by the Tukey-Kramer multiple comparison test with a level of

significance of p < 0.05 using the program Statcel2, version 2 (OMS, Tokyo,

Japan).

3. Results and discussion

3.1. Antioxidant activities of hot water extract from SRS waste for inhibiting fish

oil oxidation

The ability of hot water extract from SRS waste to prevent fish oil oxidation

was evaluated. The extract of SRS waste or leaf was incorporated at 1000 ppm

into bulk fish oil. The POV was used to measure the primary lipid-oxidation

products, especially hydroperoxides. Their inhibitory effects are shown in Fig. 1a.

As expected, as storage time increased the POV gradually increased, with the

12
highest value observed for the control (p < 0.05). The decrease in POV on day 5 in

the control could be explained by the decomposition of hydroperoxides. The POV

revealed that both hot water extracts of SRS waste and leaf significantly inhibited

peroxides formation in fish oil after 1 day (p < 0.05). The SRS extract was better

at preventing peroxides formation in fish oil than that of leaf extract after 2 days

(p < 0.05). In China, the maximum allowable POV of commercial fish oil is 20

meq kg -1. The fish oil with added SRS extract was still below this value on day 2

while the fish oil with added leaf extract and the control significantly exceeded the

allowable value (Fig. 1a).

The TBARS value gave a measure of the lipid-oxidation development in

terms of secondary oxidation products. The TBARS value increased continuously

in all samples throughout the 5 days of storage (p < 0.05) (Fig. 1b). This indicates

that the fish oil undergo oxidation during the storage. According to the TBARS

values, the important antioxidant effects could be attributed to the presence of leaf

and SRS extracts on day 1. The significant decrease of TBARS values in SRS

extract group, compared to the control group, could still be observed during day 2

to day 4 (p < 0.05). However, there was no significant difference in the TBARS

values between leaf extract group and the control group after day 2 (p > 0.05). The

inhibitory effect of SRS extract on TBARS was stronger than that of leaf extract.

It was coincidental with the finding in POV assay. Previous studies have shown

13
that the hot water extract of S. rebaudiana inhibited the oxidation of sardine oil

more effectively than green tea extract or VE (Xi et al., 1998a). VE is a well-

known antioxidant using in fish oil oxidation by the food industry. The results of

present study indicate that SRS waste can be exploited as a strong natural

antioxidant material to inhibit fish oil oxidation.

Phenolic compounds have been considered as important compounds

contributing to the antioxidant activity of stevia leaf extract (Shukla, Mehta,

Mehta, & Bajpai, 2012). To better understand the relationship between antioxidant

capacity and total phenolic content, the total phenolic contents of SRS and leaf

extracts were determined as 46.14 and 71.46 mg GAE g-1 , respectively (Fig. 1c).

The total phenolic content of the leaf extract in the present study was higher than

that extracted with water by merceration process, reaching values as high as those

extracted with ethanol in previous reports (Gawełbęben et al., 2015; Shukla,

Mehta, Bajpai, & Shukla, 2009; Shukla et al., 2012). Moreover, the method of

extraction, using only hot water as a solvent, makes it more environmentally-

friendly.

The in vitro antioxidant activities of the SRS extract were analyzed using

DPPH (alcoholic solution) and ORAC (aqueous solution) assays. The effect of

antioxidants in the DPPH assay is thought to be caused by their radical scavenging

ability or their hydrogen-donating capacity. Because this method is very sensitive

14
and simple, it has been widely used in many natural antioxidant studies (Moon &

Shibamoto, 2009). The ORAC assay, also widely used for assessing antioxidant,

evaluates the capacity of samples to scavenge peroxyl radicals, the most

physiologically significant reactive oxygen species (B. Ou et al., 2001). The

DPPH radical scavenging activities in 4 different concentrations (0.004 -0.04%) of

extracts were examined (Fig. 1d). The leaf extract exhibited significantly higher

DPPH radical scavenging activity than the SRS extract (p < 0.05). The ORAC

values of the SRS and leaf extracts were 1.15 and 1.88 µmol TE mg-1 of dry

extract, respectively (Fig. 1e). The SRS extract has a significantly lower total

phenolic content, DPPH radical scavenging activity and ORAC value than the leaf

extract (Fig. 1) (p < 0.05). In SRS and leaf extracts, the good correlation observed

between in vitro antioxidant activity and the total phenolic content suggests that

phenolic compounds are major contributors to these activities.

The oxidation of fish oil generally occurs because of free radicals generated

by light and heat (Zuta et al., 2007). The radical scavenging activity of

antioxidants can be evaluated by combining the DPPH and ORAC assays. In most

antioxidants, a positive relationship can be observed between radical scavenging

activity and the inhibition capacity of fish oil oxidation (Sekhon-Loodu et al.,

2013). However, for SRS and leaf extracts, this was not the case (Fig. 1). This

discrepancy could be explained by the relative physical locations between their

15
antioxidant ingredients and oxidative reactions (Chaiyasit, Elias, Mcclements, &

Decker, 2007). From the view of their microenvironment, fish oil is composed of

association colloids, such as reverse micelles. The physical properties of

antioxidants influence their locations in association colloids thus affecting their

efficiency when inhibiting the oxidation of the fish oil. The results of the present

study suggest that the major contributors are different in the SRS and leaf extracts,

with the primary antioxidant components of the SRS extract inhibiting the

oxidation of fish oil more effectively. Therefore, the primary antioxidant phenolic

components in the SRS extract were further analyzed through an activity-guided

fractionation approach.

3.2. Fractionation of SRS extract

After discoloration and desalting, the SRS extract was performed dialysation.

The amount of outer dialysate recovered was approximately 9.4 times that of the

inner dialysate. The DPPH radical scavenging and ORAC assays showed that the

activity of the outer-dialysate was 1.9- and 6.3-fold more effective than the inner-

dialysate, respectively (data not shown). This was based on comparing the

concentrations in the SRS extract. In the fish oil oxidation assay, the SRS outer

dialysate retained almost all the antioxidant activity of the SRS extract, whereas

the inner-dialysate showed little antioxidant activity (data not shown). Therefore

the SRS outer dialysate was further analyzed.

16
 The ODS column chromatogram of the SRS outer dialysate showed several

peaks at 250 nm and was divided into five fractions A, B, C, D and E (Fig. 2d).

The DPPH radical scavenging activities, ORAC values and percentage inhibitions

of fish oil oxidation are summarized in Fig 2. The order of DPPH radical

scavenging activities for the fractions was as follows: B > D > E ≈ A > C and for

ORAC values: B > D ≈ E ≈ C ≈ A. The POV revealed that the order of percentage

inhibitions of peroxides formation in fish oil was A > B ≈ E > C > D and for

percentage inhibitions of TBARS formation: E ≈ B ≈ C ≈ A > D. Fraction A had

no absorption peak in the UV spectrum and exhibited the highest antioxidant

activity against fish oil oxidation in POV assay, a similar finding to previous

reports (Xi et al., 1998a). This fraction contained mainly potassium which

contributed to its antioxidant activity (Xi et al., 1998a; Xi, Yamaguchi, Sato, &

Takeuchi, 1998b). Fractions B-E had absorption peaks between 250 and 350 nm,

suggesting the presence of phenolic compounds. Among them, Fractions B and E

showed the highest antioxidant activity to inhibit fish oil oxidation (Fig. 2a, 2b).

Fraction B also showed the highest radical scavenging activities (Fig. 2c, 2d).

Therefore, fractions B and E were further separated by a semi-preparative HPLC

system. The primary antioxidant phenolic compounds were purified from the

primary antioxidant activity-containing fractions and their structures are shown in

Fig. 3.

17
3.3. Identification of phenolic compounds

Compound 1, vanillic acid 4-O-β-D-glucopyranoside (Fig. 3), was obtained

from fraction B as a colorless amorphous solid. Its molecular formula, C14 H18O9 ,

was determined from the negative HR-ESI-MS spectrum which showed the

[M−H]– ion at m/z 329.0864 (calculated for C14 H17 O9, 329.0873). 1 H NMR (600

MHz, D2 O): δ 7.62 (1H, d, J = 1.8 Hz, H-2), 7.20 (1H, d, J = 9.0 Hz, H-5), 7.62

(1H, dd, J = 9.0, 1.8 Hz, H-6), 3.90 (3H, s, H-8), 5.21 (1H, d, J = 7.2 Hz, H-1ʹ),

3.61-3.64 (1H, m, H-2ʹ), 3.60 (1H, t, J = 9.6 Hz, H-3ʹ), 3.50 (1H, dd, J = 9.6, 9.0

Hz, H-4ʹ), 3.63 (1H, ddd, J = 9.0, 6.0, 2.4 Hz, H-5ʹ), 3.74 (1H, dd, J = 12.6, 6.0 Hz,

H-6ʹa), 3.89 (1H, dd, J = 12.6, 2.4 Hz, H-6ʹb). 13 C NMR (150 MHz, D2 O): δ 125.8

(C-1), 113.8 (C-2), 148.5 (C-3), 149.6 (C-4), 115.1 (C-5), 123.9 (C-6), 170.8 (C-

7), 56.2 (C-8), 100.0 (C-1ʹ), 72.9 (C-2ʹ), 75.7 (C-3ʹ), 69.5 (C-4ʹ), 76.4 (C-5ʹ), 60.6

(C-6ʹ) [Identical to data in the literature (Sakushima, Coşkun, & Maoka, 1995)].

The NOE correlations observed were 12% between H-1ʹ and H-5, and 3.5%

between H-8 and H-2. Therefore, the structure of compound 1 is determined to be

vanillic acid 4-O-β-D-glucopyranoside. Notably, vanillic acid 4-O-β-D-

glucopyranoside was found for first time in S. rebaudiana.

Compound 2, protocatechuic acid (3,4-dihydroxybenzoic acid, Fig. 3), was

obtained from fraction B as a white amorphous solid. Its molecular formula,

C7 H6O4, was determined from the negative HR-ESI-MS spectrum that showed the

18
[M−H]– ion at m/z 153.0199 (calculated for C7 H5 O4 , 153.0188). 1 H NMR (400

MHz, D2 O): δ 7.33 (1H, d, J = 2.0 Hz, H-2), 6.82 (1H, d, J = 8.4 Hz, H-5) and

7.32 (1H, dd, J = 8.4, 2.0Hz, H-6). 13 C NMR (100 MHz, D2 O): δ 123.3 (C-1),

117.3 (C-2), 143.6 (C-3), 148.3 (C-4), 115.7 (C-5), 123.2 (C-6) and 173.1 (C-7).

The structure of compound 2 was confirmed by NMR and HR-ESI-MS

spectroscopy and was consistent with the literature data for protocatechuic acid

(Jayaprakasha, Ohnishi-Kameyama, Ono, Yoshida, & Jaganmohan, 2006).

Compound 3, caffeic acid (Fig. 3), was obtained from fraction E as a yellow

crystalline powder. Its molecular formula, C9 H8 O4, was determined from the

negative HR-ESI-MS spectrum with the [M−H]– ion at m/z 179.0346 (calculated

for C9 H7 O4, 179.0344). 1 H NMR (400 MHz, D2 O): δ 7.12 (1H, d, J = 0.8 Hz, H-2),

6.89 (1H, d, J = 8.4 Hz, H-5), 7.04 (1H, dd, J = 8.4, 0.8 Hz, H-6), 7.37 (1H, d, J =

13
16.0 Hz, H-7) and 6.30 (1H, d, J = 15.6 Hz, H-8). C NMR (100 MHz, D2 O): δ

127.8 (C-1), 115.1 (C-2), 144.4 (C-3), 146.5 (C-4), 116.4 (C-5), 122.2 (C-6),

143.7 (C-7), 118.3 (C-8) and 173.7 (C-9). The detailed spectroscopic analysis data

of the compound 3 were in accordance with the literature data for caffeic acid

(Kelley, Harruff, & Carmack, 1976).

Compound 4, chlorogenic acid (5-O-caffeoylquinic acid, Fig. 3), was

obtained from the fraction E as a pale brown powder. Its molecular formula,

C16 H18 O9, was determined from the negative HR-ESI-MS spectrum which showed

19
the [M−H]– ion at m/z 353.0868 (calculated for C16 H17 O9, 353.0873). 1 H NMR

(400 MHz, D2 O): δ 1.92 (1H, m, H-2a), 2.03 (1H, dd, J = 15.2, 3.2 Hz, H-2b),

4.11 (1H, q, J = 3.2 Hz, H-3), 3.74 (1H, dd, J = 9.6, 3.2 Hz, H-4), 5.18 (1H, td, J =

9.6, 4.4 Hz, H-5), 1.91 (1H, dd, J = 13.2, 9.6 Hz, H-6a), 2.07 (1H, ddd, J = 13.2,

4.4, 2.0 Hz, H-6b), 7.02 (1H, d, J = 1.2 Hz, H-2ʹ), 6.79 (1H, d, J = 8.0 Hz, H-5ʹ),

6.95 (1H, dd, J = 8.4, 1.2 Hz, H-6ʹ), 7.47 (1H, d, J = 15.6 Hz, H-7ʹ), 6.21 (1H, d, J

= 16.0 Hz, H-8ʹ). 13C NMR (100 MHz, D2 O): δ 76.6 (C-1), 37.3 (C-2), 70.6 (C-3),

72.7 (C-4), 71.3 (C-5), 38.2 (C-6), 180.2 (C-7), 127.2 (C-1ʹ), 115.3 (C-2ʹ), 144.4

(C-3ʹ), 147.2 (C-4ʹ), 116.4 (C-5ʹ), 122.8 (C-6ʹ), 146.2 (C-7ʹ), 114.9 (C-8ʹ), 169.2

(C-9ʹ). The structure of compound 4 was identified as chlorogenic acid on the

basis of detailed NMR and HR-ESI-MS data, as well as by compared with the

literature data (Kelley et al., 1976; Morishita, Iwahashi, Osaka, & Kido, 1984).

Compound 5, cryptochlorogenic acid (4-O-caffeoylquinic acid, Fig. 3), was

obtained from the fraction E as a pale brown powder. Its molecular formula,

C16 H18 O9, was determined from the negative HR-ESI-MS spectrum with the

[M−H]– ion at m/z 353.0868 (calculated for C16 H17 O9, 353.0873), which is

isomeric to compound 4. 1 H NMR (400 MHz, D2 O): δ 1.89 (1H, ddd, J = 14.4, 4.0

1.2 Hz, H-2a), 2.04 (1H, dd, J = 14.4, 3.2 Hz, H-2b), 4.20 (1H, q, J = 3.2 Hz, H-3),

4.77 (1H, dd, J = 9.2, 2.8 Hz, H-4), 4.16 (1H, td, J = 10.0, 4.4 Hz, H-5), 1.89 (1H,

dd, J = 13.6, 10.6 Hz, H-6a), 2.04 (1H, m, H-6b), 7.05 (1H, d, J = 0.8 Hz, H-2ʹ),

20
6.79 (1H, d, J = 8.0 Hz, H-5ʹ), 6.98 (1H, dd, J = 8.0, 0.8 Hz, H-6ʹ), 7.54 (1H, d, J

= 16.0 Hz, H-7ʹ), 6.30 (1H, d, J = 16.0 Hz, H-8ʹ). 13 C NMR (100 MHz, D2 O): δ

76.4 (C-1), 37.6 (C-2), 68.2 (C-3), 77.8 (C-4), 65.0 (C-5), 40.5 (C-6), 180.5 (C-7),

127.2 (C-1ʹ), 115.4 (C-2ʹ), 144.4 (C-3ʹ), 147.2 (C-4ʹ), 116.4 (C-5ʹ), 122.9 (C-6ʹ),

146.5 (C-7ʹ), 114.7 (C-8ʹ), 169.1 (C-9ʹ). The detailed spectroscopic analysis data

of compound 5 were in accordance with the literature data for cryptochlorogenic

acid (Morishita et al., 1984; Tatefuji, Izumi, Ohta, Arai, Ikeda, & Kurimoto, 1996).

3.4. Antioxidant activities of purified compounds from SRS extract

After checking the purity of each compound using HPLC, the antioxidant

activities of these five compounds (1-5) from SRS were determined by DPPH,

ORAC and fish oil oxidation assays with an authentic standard, sodium L-

ascorbate (SA) (Fig. 4). The levels of antioxidant activity were compared at the

same molar concentration. Chlorogenic acid and cryptochlorogenic acid each

showed significantly higher DPPH radical scavenging activity than SA (p < 0.05).

Protocatechuic acid, chlorogenic acid, cryptochlorogenic acid and caffeic acid

each showed higher antioxidant activities from the ORAC and POV assays than

SA (p < 0.05). However, there was no difference among them in TBARS assay

(p > 0.05). The order of decreasing antioxidant activity against fish oil oxidation

in POV assay was: protocatechuic acid > chlorogenic acid > cryptochlorogenic

acid > caffeic acid > vanillic acid 4-O-β-D-glucopyranoside in Fig. 4a. Similar

21
results have also been found for the inhibition of soy oil oxidation (Onyeneho &

Hettiarachchy, 1992). Using automated systems for DPPH and ORAC assays,

other authors have reported similar results for caffeic acid (DPPH: 1.11 µmol TE

µmol-1 (Villaño, Fernández-Pachón, Troncoso, & García-Parrilla, 2005), ORAC:

6.63 µmol TE µmol-1 (Dávalos, Carmen Gómezcordovés, & Bartolomé, 2004)),

chlorogenic acid (DPPH: 1.04 (Morel et al., 2013), ORAC: 5.70 (Dávalos et al.,

2004)) and protocatechuic acid (DPPH: 1.29 (Villaño et al., 2005), ORAC:

5.14(Huang, Ou, Hampsch-Woodill, Flanagan, & Prior, 2002)). For ORAC assays,

the same relative order has also been reported: caffeic acid > protocatechuic acid

(Guo, Cao, Sofic, & Prior, 1997; Kayano, Kikuzaki, Fukutsuka, Mitani, &

Nakatani, 2002), caffeic acid > chlorogenic acid and cryptochlorogenic acid >

chlorogenic acid (Kayano et al., 2002), although a few differences existed

(Dávalos et al., 2004; Fernandezpanchon, Villano, Troncoso, & Garciaparrilla,

2008). Variability among results in the literature may be attributed to the use of

different equipment, analytical products and analysts.

Interestingly, among compounds 2-5, protocatechuic acid exhibited the

highest antioxidant activity to inhibit peroxides formation in fish oil, whereas it

had the lowest antioxidant activities in the DPPH and ORAC assays (Fig. 4). VE

also showed similar characteristics. In previous reports, VE has been shown to

have a dramatically lower DPPH radical scavenging activity than SA (Yu, Wang,

22
& Xi, 2008). In contrast, in the present study, VE exhibited a significantly

stronger inhibition of peroxides formation in fish oil than SA (Fig. 4a) (p < 0.05).

Moreover, they have similar antioxidant activity to inhibit TBARS formation of

fish oil (Fig. 4b) (p > 0.05). These results have further shown the importance of

the physical properties of antioxidants on inhibiting lipid oxidation.

In addition, the hot water extract of S. rebaudiana inhibited peroxides

formation of sardine oil more effectively than VE at the same mass concentration,

which can be attributed to the presence of large amounts of potassium (Xi et al.,

1998a, 1998b). Although showing significant synergistic effects with the activity

of antioxidants, potassium salts have a lower antioxidant capacity for inhibiting

peroxides formation of linoleic acid than VE at the same mass concentration (Xi et

al., 1998b). Therefore, the key factors are still ambiguous. In the present study,

protocatechuic acid, chlorogenic acid and cryptochlorogenic acid were isolated as

components contributing to the main antioxidant activity of SRS with each

component exhibiting a higher antioxidant capacity for inhibiting peroxides

formation of fish oil than VE (Fig. 4a). Our results will help to uncover the cause

of the above phenomenon.

S. rebaudiana leaf, containing a high level of phenols with strong in vitro

antioxidant activity, has attracted much attention and research (Molina-Calle,

Priego-Capote, & Luque de Castro, 2017; Periche et al., 2015; Yildiz-Ozturk et al.,

23
2015). The stems of S. rebaudiana have usually been ignored and discarded as

waste probably because of their lower levels of phenols and in vitro antioxidant

activity. The present study has investigated the exploitation of SRS waste as

antioxidants for inhibiting the oxidation of fish oil and found that the SRS extract

was more effective than the leaf extract (Fig. 1a). These results will help to

promote further research on the reuse of material once considered to have little

use. Furthermore, the primary antioxidant phenols in the SRS extract have been

isolated using an activity-guided fractionation approach when the activities were

compared at the concentration present in the SRS extract. Their antioxidant

activities were examined and interesting properties found. Although most of the

phenolic compounds identified were known components of S. rebaudiana, their

contribution to the main antioxidant capacity of the extract was little known. The

present study has also provided a basis for further research to explore the reasons

why the SRS extract is more efficient at inhibiting the oxidation of fish oil than

the leaf extract. This may contribute to discovering why the collocation of

components has a significant effect on improving the antioxidant activity of SRS

extract.

4. Conclusion

24
 In conclusion, this is the first report to find that the hot water extract of SRS

waste has higher antioxidant activity than the leaf extract for inhibiting the

oxidation of fish oil. Five phenolic compounds were isolated and identified as

major antioxidant phenolic compounds in the SRS extract, with vanillic acid 4-O-

β-D-glucopyranoside being found for the first time in S. rebaudiana. These results

have revealed that SRS could be an excellent natural source of antioxidant

materials. This increases the potential value of stem waste in terms of its

feasibility for commercial applications such as in food additives, feed additives,

health supplements and neutraceuticals.

Acknowledgements

This study was supported by the Project Sponsored by the Scientific Research

Foundation for the Returned Overseas Chinese Scholars, State Education Ministry,

Startup Project of Doctor Scientific Research in Ludong University (LY2013022),

the National Natural Science Foundation of China (No. 21502164), Research Fund

for Excellent Young and Middle-age Scientists of Shandong Province (No.

BS2015YY039), Startup Project of Doctor Scientific Research in Yantai

University (YX14B17). The authors are grateful to Prof. Minoru Ueda from

Graduate School of Science in Tohoku University, Japan for guidance and support.

25
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Figure captions

Fig. 1. Antioxidant activities and total phenolic contents of hot water extract of

Stevia rebaudiana stem (SRS). (a) Peroxide value (POV) and (b) thiobarbituric

acid-reactive substances (TBARS) values of fish oil during the conservation

period (in days) at 50 ºC. (c) Total phenolic content, (d) DPPH radical scavenging

activity and (e) ORAC value. For hot water extract of S. rebaudiana leaf (leaf)

and stem waste (stem), a final concentration of 1mg/g was used in fish oil samples.

1
Expressed as µmol of Trolox equivalents (TE) per mg of dry extract. Error bars

29
represent standard deviation (n = 3 except for ORAC assay where n = 4). Different

letters denote significant differences between mean values (p < 0.05).

Fig. 2. Antioxidant activities of SRS extract fractions as determined by (a)

percentage inhibition of POV formation and (b) TBARS formation in fish oil, (c)

DPPH radical scavenging assay, and (d) ORAC assay. (e) A column

chromatogram of the SRS extract at 250 nm. 1Expressed as µM TE per 25 mg mL-

1
of SRS extract. 2 Expressed as µM TE per 13 µg mL-1 SRS extract. Error bars

represent standard deviation (n = 3 except for ORAC assay where n = 4). Different

letters denote significant differences between mean values (p < 0.05).

Fig. 3. Structure of compounds 1-5 isolated from SRS extract. 1, vanillic acid 4-

O-β-D-glucopyranoside; 2, protocatechuic acid; 3, caffeic acid; 4, chlorogenic acid;

5, cryptochlorogenic acid.

Fig. 4. Antioxidant activities of purified compounds by (a) percentage inhibition

of POV formation and (b) TBARS formation in fish oil, (c) DPPH radical

scavenging assay, and (d) ORAC assay. VG: vanillic acid 4-O-β-D-

glucopyranoside; PA: protocatechuic acid; CH: chlorogenic acid; CR:

cryptochlorogenic acid; CA: caffeic acid; SA: sodium L-ascorbate; VE: vitamin E.

For all purified compounds, a final concentration of 1mM was used in fish oil

samples. 1Expressed as µmol TE per µmol of pure compound. Error bars represent

30
standard deviation (n = 3 except for ORAC assay where n = 4). Different letters

denote significant differences between mean values (p < 0.05).

31
Highlights
It’s the first time to find the potential for using Stevia rebaudiana stem waste.
Hot water extract of SRS has a high antioxidant activity against fish oil oxidation.
Five phenols were isolated and identified as major antioxidant phenols in SRS.
Protocatechuic acid has the highest ability to inhibit peroxide formation among
them.
Vanillic acid 4-O-β-D-glucopyranoside was isolated for the first time in Stevia.

32