180 
 

Journal homepage: www.fia.usv.ro/fiajournal 
Journal of Faculty of Food Engineering,  

Ştefan cel Mare University of Suceava, Romania  
Volume XV, Issue 2 - 2015, pag.  180 - 186 

 

 

TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF INFUSIONS 

FROM TWO BY-PRODUCTS: WALNUT (JUGLANS REGIA L.) SHELL AND 

ONION (ALLIUM CEPA L.) 

*Sonia AMARIEI1, Valentina MANCAS1 

1Faculty of Food Engineering, Stefan cel Mare University of Suceava, Romania 
gutts@fia.usv.ro 

*Corresponding Author 
Received 25th April 2016, accepted 27th June 2016 

 
 

Abstract: Total phenolic content and antioxidant activity of two by-products of onion (Allium cepa L.) 
and walnut (Juglans regia L.) processing were studied in order to establish the possibility of 
capitalization as antioxidant sources. The by-products analyzed were walnut shell and onion peel. 
From these by-products, infusions were prepared using water as solvent. Total phenolic content was 
determined using Folin-Ciocalteu method, while the radical scavenging ability of the infusions was 
monitored using the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl). Walnut shell infusion 
had a relatively low content in phenolic compounds (32.08 mg GAE/g), below the level reported for 
other walnut by-products. Given the low phenolic content, walnut shell infusion exhibited a reduced 
radical scavenging capacity (7.82%). On the contrary, the onion peel infusion had both high phenolic 
content (280.32 mg GAE/g) and antioxidant activity (69.38%). Based on these two results it was 
confirmed that onion peels resulted from bulb processing are suitable for reuse in antioxidants’ 
extraction in both food and pharmaceutical industries. 
 
Keywords: walnut, onion, antioxidants, DPPH, Folin-Ciocalteu 
 
 
1. Introduction 
 
Official surveys indicate that the European 
food-processing industry produces vast 
volumes of wastes every year, and despite 
the efforts to reduce solid waste volume the 
impact of food industry on the environment 
remains unchanged. Apart from the 
environmental challenges posed, such 
streams represent considerable amounts of 
potentially reusable materials and energy 
[1]. Fruit and vegetable processing has the 
highest wastage rate of any food, rising up 
to 16.4% for fruits and to 25.8% in the case 
of vegetables [2]. According to Food and 
Agriculture Organization (FAO), nearly 
50% of all fruits and vegetables in the EU 
go to waste, 5.5% only being lost during 

postharvest handling and processing [3]. 
Byproducts discharged from manufacturing 
of fruit or vegetable-based products depend 
on the fresh produce used and include peels, 
skin, hulls, pips, and cores. Fruit and 
vegetable waste can be capitalized in 
added-value products such as animal feed, 
single-cell protein (SCP) and other 
fermented products, baker’s yeast, organic 

acids, amino acids, enzymes (e.g. lipases, 
amylases, and cellulases), flavors and 
pigments, gums and polysaccharides [4,5]. 
Walnut (Juglans regia L.) is a tree native to 
a region stretching from the Balkans 
eastward to the western Himalayan chain 
[6,7]. The walnut is traditionally cultivated 
throughout Europe for its valuable wood 
and seeds (walnuts). Seeds are of high 

http://www.fia.usv.ro/fiajournal
mailto:gutts@fia.usv.ro


Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XV, Issue 2  – 2016 

Sonia AMARIEI, Valentina MANCAS, Total phenolic content and antioxidant activity of infusions from two by-products: 
walnut (juglans regia l.) shell and onion (allium cepa l.), Food and Environment Safety, volume XV, Issue 2 – 2016, pag. 
180 - 186 
 
 

181 

economic interest to the food industry and 
are valued globally for the nutritional, 
health and sensory attributes [8]. Romania 
is among the first ten world producers of 
walnut. In order to maximize the 
productivity and reduce the waste from 
processing, several by-products derived 
from the walnut tree, such as green walnuts, 
shells, kernels, bark, green walnut husks, 
and leaves have been used in both cosmetic 
and pharmaceutical industries [9,10]. 
Walnut shells and green walnut husk have 
various possible uses in the industry. 
Recent research has reported on the great 
potential of green walnut husk to remove 
Lanaset Red G from aqueous solution [11]. 
It was also investigated the ability of a 
powdered activated carbon (PAC) derived 
from walnut shell to adsorb mercury from 
aqueous solutions [12,13], and the reuse of 
walnut shell as a support material 
(biosorbent) for effective cesium adsorption 
from aqueous solution [14]. In addition to 
non-food applications, the central point of 
recent research is the phenolic composition 
and antioxidant activity of extracts from by-
products. While several studies reported on 
the bioactive compounds of extracts from 
green walnut husk [10, 15-17], no data is 
available on the composition of aqueous 
extracts from walnut shells. 
Onion (Allium cepa L.) is one of the major 
vegetable crops grown worldwide, after 
potato and tomato, with a production that 
was estimated to 84.75 Mt in 2013 [18] and 
increases constantly. Onions possess strong, 
characteristic aromas and flavours [19] that 
made the vegetable (bulb or scallion) an 
important ingredient of food. On account of 
the whopping production and use of onion, 
bulbs and scallions processing raises some 
concern over the large quantity of industrial 
waste produced and its disposal. 
Furthermore, the perishable nature make 
onion waste not suitable for fodder or 
landfill disposal due to the rapid growth of 
phytopathogens, e.g. Sclerotium cepivorum 
(white rot) [20].  

In these conditions, the only suitable 
decision in onion waste management is the 
capitalization of by-products. The main 
onion by-products include onion skins 
generated during industrial peeling, and 
undersized, malformed or damaged bulbs 
[21]. Onion peel importance in bioactive 
compounds extraction is given by its high 
levels of two chemical groups with proven 
health benefits for humans, namely 
flavonoids and alk(en)yl cysteine sulfoxides 
(ACSO) [22]. The main flavonoid present 
in onion peel is quercetin aglycon [23], a 
compound known for its antioxidant and 
free radical scavenging activity, 
cardioprotective effect, anticancer, 
antiinflamatory and antiviral activity, as 
well as inhibitory activity against bacterial 
ureases [24-26]. Naturally occurring 
ASCOs in onion peel are trans-(+)-S-(1-
propenyl)-L-cysteine sulphoxide, (+)-S-
methyl-L-cysteine sulphoxide, and (+)-S-
propyl-L-cysteine of sulphoxide [27]. 
Although Romania has a high production of 
walnuts and onion, the capitalization of 
waste resulted from processing has been 
less studied. The aim of this work was to 
analyze the composition in antioxidants of 
infusions from walnut shells, and onion 
peel in order to establish the possibility of 
reuse in Romanian industry according to the 
by-product composition. The solvent used 
in the extraction process was water. No 
alcoholic solvent was added to enhance the 
extraction. The parameters analyzed were 
total phenolic content and scavenging effect 
on DPPH (2,2-diphenyl-1-picrylhydrazyl). 
 
2. Matherials and methods 
 
2.1. Plant material 
 
Onions were purchased from an organic 
farmer located in Suceava County, in north-
eastern Romania. The bulbs were from the 
2015 harvest. Walnuts were collected in 
September 2015 from a single tree that had 
not received any insecticide treatment.  



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XV, Issue 2  – 2016 

Sonia AMARIEI, Valentina MANCAS, Total phenolic content and antioxidant activity of infusions from two by-products: 
walnut (juglans regia l.) shell and onion (allium cepa l.), Food and Environment Safety, volume XV, Issue 2 – 2016, pag. 
180 - 186 
 
 

182 

The walnut shell was cracked and the 
kernel was removed, while the outer skin of 
the onion was sectioned, and then peeled. 
Both by-products were rinsed prior to being 
used. To enhance the extraction of bioactive 
compounds onion peels were cut into 
chunks and walnut shells were crushed. 
 
2.2. Preparation of individual infusions 
 
The infusion (Fig. 1) was prepared as 
follows: 80 g of freshly separated onion 
peels or walnut shells was placed in a 
stainless steel pot and 100 ml of water 
brought to the boiling point (100 °C) was 
added. The pot was quickly covered leaving 
the mix to infuse for about 15 minutes. The 
cover was then removed and the content 
was allowed to rest for about 4 hours. 
Finally, peels and shells were retained in a 
filter and the infusion was stored in a closed 
container until its composition was 
analyzed. 
 
2.3. Total phenolic content determination 
 
Total phenolic content of the obtained 
infusions was determined colorimetrically 
using the method described by Singleton 
and Rossi (1965) with some modifications 
[28]. Briefly, 50 µl of infusion was mixed 
with 250 µl of Folin-Ciocalteu reagent. 
After 3 minutes 500 µl of saturated sodium 
carbonate solution (20%, w/w) was added 
and mixed again. The mixture was allowed 
to stand for 30 minutes in the dark prior to 
measuring the absorbance at 765 nm in a 
single beam UV-Vis-NIR spectro-

photometer (Shimadzu Corporation, Japan). 
Total phenolic content was expressed in mg 
gallic acid equivalents (GAE)/g. For gallic 
acid, absorbance is described by the 
following equation: 

y = 0.0012x – 0.0345 (R2 = 0.9997) 
 

2.4. Antioxidant activity 
 
The radical scavenging ability of the 
infusions was monitored using the stable 
free radical DPPH (2,2-diphenyl-1-
picrylhydrazyl) following a procedure 
similar to those proposed by Mraicha et al. 
(2010) and Bouaziz et al. (2008) in previous 
studies [29,30]. Aqueous solutions of 
samples were prepared by mixing various 
concentration of infusion with ethanol 
solution, as follows: 60 µl onion peel 
infusion and 2940 µl solvent, and 30 µl 
walnut shell infusion in 2970 µl ethanol. 
The aliquots were added to 5 ml of a 
0.004% (w/w) freshly prepared methanol 
solution of DPPH. The mixtures were left to 
stand at room temperature for 60 minutes in 
the dark (until stable absorbance values 
were obtained). The absorbance was 
measured against a blank at 517 nm in a 
single beam UV-Vis-NIR spectro-
photometer (Shimadzu Corporation, Japan). 
Free radicals inhibition (I%) was calculated 
using the equation: 
I% = [(Ablank – Asample)/Ablank] ×100, 
Where: Ablank – absorbance of the control 
reaction, containing all the reagents except 
the analyzed infusion 
Asample – absorbance of the infusion 

 
Fig. 1 Walnut shell infusion (left) and onion peel infusion (right) 

 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XV, Issue 2  – 2016 

Sonia AMARIEI, Valentina MANCAS, Total phenolic content and antioxidant activity of infusions from two by-products: 
walnut (juglans regia l.) shell and onion (allium cepa l.), Food and Environment Safety, volume XV, Issue 2 – 2016, pag. 
180 - 186 
 
 

183 

 
Fig. 2 Absorbance (left) and total phenolic content (right) of infusions 

 
 

3. Results and discussion 
 
3.1. Total phenolic content of infusions 
 
The Folin-Ciocalteu assay, used for the 
determination of the total phenolic content 
of infusions from walnut shells and onion 
peel is a simple and widespread method that 
has been employed for a long time as a 
measure to evaluate total phenols of natural 
products. Extraction of phenolic 
compounds from plant material is 
influenced by the chemical nature of the 
compound, the extraction method 
employed, sample particle size, the solvent 
used, extraction conditions (time, 
temperature), as well as the presence of 
interfering substances [31]. Unlike the 
extraction with alcoholic solvents 
(methanol, ethanol), the use of water in the 
solubilization of bioactive compounds does 
not rise concern regarding the toxicity and 
recovery of the solvent from the extract. 
Recent studies on the extraction of 
anthocyanins, carried out on synthetic 
flavylium salts showed that the molecules 
require neutralizing their own electrostatic 
repulsions with water molecules so that the 
dimerisation can be carried out [32]. 
Therefore, water plays a fundamental role 
in phenolic compounds extraction. 

Total phenolic content of walnut shell 
infusion and onion peel infusion, alongside 
the absorbance measured is presented in 
Fig. 2. As it was expected, total phenolic 
content of walnut shell infusion (32.08 mg 
GAE/g) was relatively low but exceeded the 
mean of 18.04 mg GAE/g reported for the 
phenolic content of methanolic extract from 
powdered walnut hull [33]. Higher phenolic 
content was determined for extracts from 
green walnut husk (50.18 mg GAE/g), 
leaves (94.39 mg GAE/g), and kernel 
(116.22 mg GAE/g) [34]. For onion peel 
infusion, total phenolic content measured 
was 280.32 mg GAE/g, a level that was 
close to the content obtained by Boo et al. 
(2012) for water extracts from powdered 
onion peel [35]. Regarding the influence of 
plant section and growth on the phenolic 
content of onion, it was noted that the 
greenish part of the onion had a higher 
phenolic content in comparison with the 
white part of the bulb [36]. 
 
3.2. Antioxidant activity 
 
Antioxidants are essential to preserve the 
biological system from free radicals 
damage to biological molecules [37].  
 
 
 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XV, Issue 2  – 2016 

Sonia AMARIEI, Valentina MANCAS, Total phenolic content and antioxidant activity of infusions from two by-products: 
walnut (juglans regia l.) shell and onion (allium cepa l.), Food and Environment Safety, volume XV, Issue 2 – 2016, pag. 
180 - 186 
 
 

184 

 
Fig. 3 Absorbance (left) and scavenging effect (right) of infusions 

 
The capacity of antioxidants to protect a 
biological system against the harmful 
effects of oxidative processes is defined as 
antioxidant activity. In this study, the ability 
of the investigated walnut shell infusion and 
onion peel infusion to act as donors of 
hydrogen atoms or electrons in DPPH 
radical transformation into its reduced form 
of DPPH•-H was investigated in DPPH• 
assay. As Fig. 3 shows, both samples 
exhibited a radical scavenging activity 
below 70%. For walnut shell infusion the 
scavenging activity measured was 7.82%, a 
value that was greater than an inhibition of 
7.19%, reported by other authors for the 
same by-product [33]. Compared to other 
by-products of walnut processing, the 
scavenging activity on DPPH radicals of 
infusions from shells was lower than the 
one obtained for water:ethanol extracts 
from green walnut husk [38]. 
Onion peel infusion, which had the highest 
content of phenols, exhibited a high DPPH 
free radical scavenging capacity, namely 
69.38%. This value is close to the 
antioxidant activity of 69.7%, measured for 
methanolic extracts from red onion peel 
[39]. As in the case of polyphenols, the 
antioxidant activity is influenced by the 
distribution of the analyzed segment in 
vegetable material. It was reported that 

anthocyanins, a group of compounds with 
high antioxidant potential are heavily 
concentrated in onion skin [40]. Moreover, 
it was observed that peel shredding led to 
an increase in the content of quercetin 4'-O-
β-glucopyranoside [41], a flavonoid with 
documented radical scavenging activity. 
These two last observations confirm the 
increase in antioxidant activity from the 
inside to the outer part (peel) of the bulb. 
 
4. Conclusion 
 
The present study reports on total phenolic 
content and antioxidant activity of two 
infusions prepared with by-products of 
onion and walnut processing. The by-
products used were walnut shells and onion 
peels, and the solvent of extraction was 
water. 
Walnut shell infusion had a low content of 
phenolic compounds, and therefore 
exhibited free radicals scavenging activity 
close to the levels reported in other similar 
studies. On the other hand, onion peel 
infusion presented a balanced phenolic 
content, and increased radical scavenging 
activity. Between the two by-products 
investigated, onion peel has a great 
potential for capitalization. Given the high 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XV, Issue 2  – 2016 

Sonia AMARIEI, Valentina MANCAS, Total phenolic content and antioxidant activity of infusions from two by-products: 
walnut (juglans regia l.) shell and onion (allium cepa l.), Food and Environment Safety, volume XV, Issue 2 – 2016, pag. 
180 - 186 
 
 

185 

content of antioxidants, the reuse could 
imply both food and non-food applications. 
 
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Volume XV, Issue 2  – 2016 

Sonia AMARIEI, Valentina MANCAS, Total phenolic content and antioxidant activity of infusions from two by-products: 
walnut (juglans regia l.) shell and onion (allium cepa l.), Food and Environment Safety, volume XV, Issue 2 – 2016, pag. 
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	1. Introduction