Comparative Study on Antioxidant Properties of Various Solvent
Extracts Relating to Indocalamus tessellatus Leaves of Agricultural
Tourism Products
Shenglang Jin
1
and Fei Que
2
1
Tourism College, Huangshan University, Anhui, Huangshan, 245041, China
2
Zhejiang Institute of Economics and Trade, Hhangzhou, 310018, China
Keywords: Indocalamus tessellatus Leaves, Ethanol, Petroleum Ether, 2-Diphenyl-1-Pyridyl Hydrazide Scavenging
Activity, Antioxidant Activity.
Abstract: The rapid development of rural tourism in China has become an important force in China's tourism industry.
Rural tourism is increasingly becoming an effective carrier to solve the problem of urban-rural integration
and the construction of new countryside, and the production, development and marketing of agricultural
products are gradually becoming a key selling point of rural tourism. As a tourist agricultural product,
Indocalamus tessellatus leaves were extracted by ethanol, petroleum ether and water respectively. Three
different extracts revealed different results of each experiment. The ethanol extract with the most total phenol
content is the most effective antioxidant in all experiments. The effects of five different pH values on the
antioxidant activities at two diverse temperatures were also studied. The antioxidant activity of the extract
kept the temperature at 40 ℃. It reached the maximum value at pH 7, and decreased gradually after hiding at
5 ℃ for 30 days (p<0.05). The results showed that Indocalamus leaf has significant antioxidant activity, so it
can be used as a potential source of natural antioxidants in the food industry.
1
INTRODUCTION
China has the most abundant bamboo resources in the
world, and the chemical components of bamboo
leaves are widely used in the production of new
medicines and foods. Indocalamus tessellatus leaf, as
one of the types of columnar leaf, has been used for
hundreds of years because of its good leaf use, long-
lasting fragrance and health care function.
Indocalamus tessellatus leaf is one of the agricultural
tourism products (Yildirim 2000). With the
deepening of people's understanding of Indocalamus
tessellatus leaf and its medicinal value, the research
and development of Indocalamus tessellatus leaf
extract have been extensively investigated in recent
years.Indocalamus tessellatus leaf has the medicinal
characteristics of diuresis, hypoglycemia and
hypotension (Blois 1958). However, until recently, it
was found that their mechanism of action was related
to antioxidant activity. Some papers reported the
content of Cathaya argyrophylla, Paeonia,
Cimicifuga nanchuanensis and other flavonoids in
Indocalamus tessellatus leaf. Phenolic compound is
the main phytochemical substance of the antioxidant
activity of agricultural tourism products. On the basis
of relevant experts and scholars' research on rural
tourism and agricultural products, this paper will
make a special study on the function improvement of
agricultural products in rural tourism, and put forward
the idea of increasing added value of agricultural
products, so as to further adjust the rural industrial
structure and provide theoretical support and policy
suggestions for increasing farmers' income
(Ohkawa
1979).
2
MATERIALS AND METHODS
2.1 Chemicals
The reference standard of rutin was purchased from
National Institute for the Control of Pharmaceutical
and Biological Products (purity ≥ 98%). 2-diphenyl-
1-pyridyl hydrazide and 2-thiobarbituric acid were
purchased from Nanjing Jiancheng Bioengineering
Institute (China). Ethanol, sodiumnitrite,
Jin, S. and Que, F.
Comparative Study on Antioxidant Properties of Various Solvent Extracts Relating to Indocalamus tessellatus Leaves of Agricultural Tourism Products.
DOI: 10.5220/0011382400003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1195-1200
ISBN: 978-989-758-595-1
Copyright
c
2022 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
1195
aluminum nitrate, sodium hydroxide were purchased
from commercial sources.
2.2 Materials
Indocalamus tessellatus leaves (chequer-shaped
tessellatus leaves, collection time: December, 2018
and July, 2019 respectively, place: Huangshan,
China). The Indocalamus tessellatus leaves were
grounded in a pulverizer and passed through an 60-
mesh sieve, dried to constant mass in the oven at
60 ℃ for 6 h, cooled and then stored.
2.3 The Effective Components
Extracted from Indocalamus
Tessellatus Leaves
The Indocalamus tessellatus leaves (20 g) were
extracted overnight with 100 ml each of ether, ethanol
or water, respectively, in a mechanical shaker at room
temperature. Each extract was filtered with filter
paper. The filtrate obtained from ether and ethanol
was evaporated to dryness at 40℃ in a rotary
evaporator (Beijing Chemical-Regent Company,
China) and the water extract was freeze-dried. The
dried sample of each extract was weighed to
determine the yield of soluble constituents and stored
at 5℃ until use.
2.4 Reducing Power Assay
Yildirim, Mavi and Kara (2000) methods were used
to evaluate the ability of extract to reduce iron (III).
The dry extract (125–1000 µg) was placed in 1 ml of
the corresponding solvent, mixed with 2.5 ml of
phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of
potassium ferricyanide (K
3
Fe (CN)
6
; 10g l
-1
), and the
mixture was incubated at 50 ℃ for 30 minutes. After
incubation, add 2.5ml trichloroacetic acid (100 g l
-1
)
and centrifugate the mixture to 1650g for 10min.
Finally, 2.5ml supernatant was mixed with 2.5ml
distilled water and 0.5ml FeCl
3
(1 g l
-1
) (Siddhuraju
2002).
2.5 2-Diphenyl-1-Pyridyl Hydrazide
Radical Scavenging Activity
Blois (1958) method was used to determine the DPPH
free radical scavenging ability of the extract. Mix 1
ml of 1 mM DPPH ether solution with 3 ml of ether
extract containing 50-400 µg of dry extract. Then
rotate the mixture vigorously and leave it in darkness
at room temperature for 30 minutes. The absorbance
was measured at 517nm and the activity was
expressed as a percentage of DPPH clearance relative
to the control using the following equation:
2-diphenyl-1-pyridyl hydrazide scavenging activity (%)
=
100
controlofAbsorbance
sample of Absorbance - control of Absorbance
×
2.6 Antioxidant Activity
The antioxidant activity of BHT was estimated. The
obtained reaction solution (1 ml) was used for 2-
thiobarbituric acid determination (Yen 1993).
The oxidation degree of oil was determined by 2-
thiobarbituric acid reported by Ohkawa, ohishi and
Yagi (1979). The above reaction solution (1ml) was
mingled with zero point two percent (w/V) 2-
thiobarbituric acid solution (3 ml) and 0.05 M sulfuric
acid (2.5 ml). Heat the mixed liquor in a 95 ℃ water
bath for 30 minutes. Cooling the solution in ice for
five minutes, 4.0 ml 1-butanol was used to extract the
colored substance. The absorbance of n-butanol layer
was measured at 532 nm. Taking malondialdehyde
diethylacetal as the standard, the standard curve was
established and expressed as malondialdehyde
equivalent. The definition of antioxidant activity
(AOA) can be expressed as the percentage of
inhibition of lipid peroxidation relative to the control
group selected in the experiment. The following
formula can be used:
AOA(%) =
Absorbance of control - Absorbance of sample
Absorbance of control
× 100
3
RESULTS AND DISCUSSION
3.1 Extract Yield and Total Phenolics
As shown in Table 1, the yield and total phenol data
of different extracts of Indocalamus tessellatus leaf.
Dry leaf weight ranges from 7.02% (water extraction)
to 13.46% (ethanol extraction). According to the
experimental results, the amount of total phenol
(gallic acid equivalent) represented as a percentage by
weight of dry extract is between 6.75% of water
extract and 8.97% of ether extract. Ether is the most
effective solvent for extraction of Indocalamus
tessellatus leaf antioxidant.
3.2 Reducing Power
Many researches had shown that the electron
donating ability of bioactive compounds is related to
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
1196
their antioxidant activity. In this paper, the ability of
the extract to reduce iron (III) to iron (II) was
determined and compared with vitamin C. All the
three extracts showed a certain degree of electron
delivery capacity, which was concentration
dependent, but the electron delivery capacity was
lower than that of vitamin C (Fig. 1, table 2). The
ether extract with the most total phenol content is the
most effective reducing agent, while the water extract
with the lowest phenol content has the weakest
property. A similar relationship between iron (III)
reduction activity and total phenol content has been
reviewed on some studies (Zhu 2002).
Table 1: Study on the content of total phenols and the extract extracted by different solvents from Indocalamus tessellatus
leaves.
Sample Yield
Total phenolics
ethanol 13.46±1.33
a
8.97±0.09
a
Petroleum ether 7.98±0.88
b
8.68±0.39
b
Water 7.02±0.97
c
6.75±0.42
c
Figure 1: Reducing powers of different solvents extracts of Indocalamus tessellatus leaves.
Figure 2: The results of different solvents extracts of Indocalamus tessellatus leaves.
Comparative Study on Antioxidant Properties of Various Solvent Extracts Relating to Indocalamus tessellatus Leaves of Agricultural
Tourism Products
1197
Table 2: Comparison of antioxidant properties of Indocalamus tessellatus leaves extracts.
Sample Reducing power TAOC
Antioxidant
activity
‡B
2-diphenyl-1-pyridyl
hydrazide EC50
BHT nd 3.873±0.026
a
86.42±0.52
a
39.11±0.48
c
Ascorbic acid 2.667±0.002
a
nd nd 58.86±0.28
b
Ethanol 0.244±0.001
b
1.385±0.004
b
79.79±0.48
b
80.49±0.79
a
petroleum ether 0.215±0.007
c
1.377±0.003
b
60.51±0.92
c
nd
Water 0.162±0.005
d
0.652±0.003
c
40.12±0.48
d
nd
Figure 3: 2-diphenyl-1-pyridyl hydrazide radical scavenging activities of ethanol, petroleum ether and water extracts of
Indocalamus tessellatus leaves.
3.3 2-Diphenyl-1-Pyridyl Hydrazide
Radical Scavenging Activity
The effect of three extracts on 2-diphenyl-1-pyridyl
hydrazide scavenging activity at different
concentrations (Fig. 3). The ether extract with the
highest total phenol content is the most active free
radical scavenger, followed by ethanol and water
extract. However, ether extract was not as effective as
BHT and ascorbic acid in the positive control group
because the amount of extract (p<0.05) required to
scavenge 50% 2-diphenyl-1-pyridyl hydrazide
radical in the reaction mixture (EC50) was
significantly higher than BHT and ascorbic acid
(Table 2). The results of 2-diphenyl-1-pyridyl
hydrazide free radical scavenging experiments show
that extracts donated by hydrogen and / or electron
may prevent the active free radical species from
reaching the biomolecules in susceptible organisms
and food systems.
3.4 Antioxidant Activity of
Indocalamus Tessellatus Leaves
Extract
In this study, 2-thiobarbituric acid was used to
determine the inhibitory effect of Indocalamus
tessellatus leaves extract on the lipid peroxidation of
flaxseed. By measuring the absorbance of
Indocalamus tessellatus leaves extract at 532 nm, the
content of thiobarbituric acid reactant produced by
Indocalamus tessellatus leaves extract on the
oxidation of flaxseed oil was determined. All three
extracts can prevent the formation of thiobarbituric
acid reactant produced by ferrous sulfate (Table 3).
According to the evaluated percentage of antioxidant
activity shown in Table 2, it is found that the extract
with the highest activity is ether extract, with the
activity of 81%, which is lower than BHT value
(90%) (p<0.05). The active sequence was related to
the total phenol content of each extract.
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
1198
Table 3: Effect of Indocalamus tessellatus extracts on the production of malondialdehyde.
Control
BHT Ethanol Petroleum ether Water
12.5ppm 25ppm 50ppm 12.5ppm 25ppm 50ppm 12.5ppm 25ppm 50ppm
6.3 0.3 2.8 1.9 0.7 3.9 3.3 2.1 5.8 5.2 3.7
malondialdehyde equivalents (mmol/kg oil)
3.5 The Influence Factors of
Temperature, Ph Value and
Stability
Table 4 shows the effect of temperature on the
antioxidant stability of ethanol extract. At fifty
degrees Celsius for sixty minutes, the antioxidant
activity value of the extract was basically the same,
and the change of malondialdehyde equivalent of oil
oxidation was not significant (p<0.05). When heated
at fifty degrees Celsius for 120 min, the antioxidant
activity of the extract decreased by 3%. When heated
at one hundred for sixty minutes, the antioxidant
activity of the extract decreased by 9% (p<0.05), and
decreased continuously with the prolongation of
boiling time. However, even after heating at One
hundred degrees Celsius for One hundred and twenty
minutes, the remaining antioxidant activity was about
sixty-eight percent. The decrease of antioxidant
activity and subsequent heating at One hundred
degrees Celsius may be related to the loss of natural
antioxidants in the extract or the formation of new
compounds with oxidation promoting activity.
The effect of pH value on the sturdiness of ether
extract is demonstrated in Table 5. In the presence of
the extraction solution, the malondialdehyde
equivalent formed by the oxidation of the oil
decreased gradually, the lowest one was at median
pH, and increased continuously at alkaline pH
(p<0.05), indicating that the antioxidant activity of
the extraction solution had a strong dependence on
the pH value of the system. The effect of storage on
the stability of Indocalamus tessellatus leaf ether
extract was also studied for more than three months
with an interval of thirty days (Table 6). There was no
change in antioxidant activity of the temperature of
5℃ for thirty days. At the end of storage, the residual
activity of the extract was about 65%, indicating that
it could still be used as a source of natural
antioxidants.
Table 4: The results of ethanolic extract.
Temperature 40℃ 80℃
Non-treated 0.48±0.02b 0.48±0.02c*
60min 0.51±0.03b 1.1+0.04b*
120min 0.73±0.03a 1.42±0.03a*
Table 5: Effect of Indocalamus tessellatus extracts on the production of malondialdehyde.
Control BHT
Ethanol Petroleum ether Water
12.5ppm 25ppm 50ppm 12.5ppm 25ppm 50ppm 12.5ppm 25ppm 50ppm
6.3 0.3 2.8 1.9 0.7 3.9 3.3 2.1 5.8 5.2 3.7
malondialdehyde equivalents (mmol/kg oil)
Table 6: The results of ethanolic extract.
PH
Malondialdehyde
equivalents
(mmol/kg oil)
3 5 7 9 11
0.536±0.02b 0.528±0.05b 0.477±0.03c 0.610±0.03b 0.698±0.04a
Comparative Study on Antioxidant Properties of Various Solvent Extracts Relating to Indocalamus tessellatus Leaves of Agricultural
Tourism Products
1199
4
CONCLUSIONS
Based on the comprehensive development status and
characteristics of rural tourism, this paper studies the
current status and problems of rural tourism
agricultural products processing, and takes
Indocalamus tessellatus leaves as an example for
analysis, proposes corresponding finishing methods,
and puts forward countermeasures and suggestions.
The possible innovation of this article is that the
subject combines rural tourism and agricultural
product processing, and proposes a new model that
uses rural tourism as a platform to enhance the deep
processing of agricultural products. It provides basic
research data for the study of antioxidant activity of
plant extracts. However, how to implement these
strategies and achieve effective results in
combination with the characteristics of agricultural
tourism products still needs further research. The
next step will be further research on other plants to
improve the scientific research level of food
processing industry.
ACKNOWLEDGEMENTS
This work is supported by 2019 Anhui Provincial
Academic Exchange Program for Excellent Young
Talents in Colleges and Universities (NO.
gxgwfx2019053),2019 provincial first-class
professional project cooking and nutrition education
(NO. 126), 2019 Anhui Provincial Quality
Engineering Project (NO.2019rcsfjd077) and 2017
Anhui Research Center of Simulation Design and
Modern Manufacturing Engineering Technology
(NO. SGCZXZD01).
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