Comparative Study on Antioxidant Properties of Various Solvent

Extracts Relating to Indocalamus tessellatus Leaves of Agricultural

Tourism Products

Shenglang Jin

and Fei Que

Tourism College, Huangshan University, Anhui, Huangshan, 245041, China

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.

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).

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

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

Fe (CN)

; 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

(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

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

8.97±0.09

Petroleum ether 7.98±0.88

8.68±0.39

Water 7.02±0.97

6.75±0.42

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

86.42±0.52

39.11±0.48

Ascorbic acid 2.667±0.002

nd nd 58.86±0.28

Ethanol 0.244±0.001

1.385±0.004

79.79±0.48

80.49±0.79

petroleum ether 0.215±0.007

1.377±0.003

60.51±0.92

Water 0.162±0.005

0.652±0.003

40.12±0.48

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.

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

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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