Comparison of Antioxidant Activity between Turmeric- Tamarind

Infused Water and Traditional Herbal using DPPH Method

Indah Tri Lestari, Amal Fadholah and Dhia Suhailah

Pharmacy Department, University of Darussalam Gontor, Siman Km 6, Ponorogo, East Java, Indonesia

Keywords: Antioxidant Activity, Turmeric-Tamarind, Infused Water, Traditional Herbal.

Abstract: Recently, functional foods and beverages have been many advances, some of which are traditional herbal and

infused water. The traditional herbal is a product of traditional Indonesian ingredients that have been

developed in the community and used from generation to generation. Meanwhile, the infused water is water

in which there are pieces of fruits, vegetables, or spices so that the water has a distinctive aroma, flavour, and

colour according to the ingredients infused in the water. The efficacy of using turmeric-tamarind infused water

and traditional herbal still largely based on empirical data. Several studies related to the antioxidant activity

of turmeric- tamarind infused water and traditional herbal are still limited. This study aimed to determine the

antioxidant activity of turmeric tamarind in infused water and traditional herbal products using the DPPH

method. The percentage of inhibition and IC50 were measured. The results showed that turmeric-tamarind

traditional herbal was known to be a strong antioxidant (IC50 value of 94.21 ppm), and turmeric-tamarind

infused water was included in the moderate antioxidant group (IC50 value 142,08 ppm). Based on the

statistical test, it can be concluded that there was a significant difference in IC50 value between turmeric-

tamarind traditional herb and turmeric-tamarind infused water.

1 INTRODUCTION

The presence of a new trend related to functional

drinks shows the high public awareness regarding

body health. Several types of functional drinks that

are well known in Indonesia are infused water and

traditional herb. Traditional herb is a product of

traditional Indonesian ingredients that have been

developed in the community and used from

generation to generation (A'yunin, 2019). Traditional

herbs are generally referred to by the name of jamu.

The names of these jamu are the main components in

the herbal ingredients, such as jamu kunir asem, then

the components are turmeric and tamarind. The

observations found that the ingredients used to make

the herbs came from nature without synthetic

chemicals, so that the herbs can be consumed

regularly and there are relatively no side effects

(Sumarni, 2019).

Different from traditional herb, infused water is

water in which there are pieces of fruits, vegetables

or spices so that the water has a distinctive aroma,

flavor and color according to the ingredients infused

in the water (Muaris, 2014).

Infused water is different from juice, because it

does not use added sugar or other additives so that

infused water is more natural for consumption

(Harifah, 2017). Infused water can be a

recommendation for those who don't like to consume

water or mineral water because of the taste and aroma

of the fruit (Muzaifa, 2019). In addition, the infused

water works as a toxin neutralizer in the body and is

very suitable for daily consumption to replace

drinking water. Consuming infused water can help

maintain health (Aprilia, 2014).

The fruit used in making functional drinks, both

in herbal medicine and in infused water, mainly

contains vitamin C which is good for maintaining the

immune system and has antioxidant activity that is

able to ward off free radicals in the body. (Harifah,

2017). Antioxidants are compounds that give

electrons to unpaired free radicals. Antioxidants are

believed to play a very important role in body defense

and protect cells from damage caused by free radicals.

Antioxidants have been investigated for the

prevention of diseases such as cancer, coronary heart

disease, and even altitude sickness (Yadav et al.

2016). Antioxidant activity in beverages can be

influenced by several factors including: Variations of

Lestari, I., Fadholah, A. and Suhailah, D.

Comparison of Antioxidant Activity between Turmeric- Tamarind Infused Water and Traditional Herbal using DPPH Method.

DOI: 10.5220/0010756400003235

In Proceedings of the 3rd International Conference on Social Determinants of Health (ICSDH 2021), pages 227-233

ISBN: 978-989-758-542-5

227

fruits, stepping time, temperature, and particle size

(Castiglioni, 2015).

Tamarind and turmeric are examples of plants that

are known to have high antioxidant activity and can

be used in various types of functional drinks. The

antioxidant activity of dry turmeric rhizomes is

known to have high antioxidant activity with an IC50

value of 46.7686 g/m (Pratiwi and Wardaniati, 2019).

Meanwhile, the antioxidant activity in tamarind fruits

extracts expressed in percentage ranged between

29.27±0.06% and 40.11±0.03% (Mbunde, 2018)

Antioxidant activity can be measured by various

methods. One of the methods is the radical scavenger

DPPH (1,1-diphenyl-2-picrylhydrazyl). DPPH

method is the most widely reported method for

screening antioxidant activity because this method is

simplest, fast, sensitive, accurate. Antioxidant

activity is expressed in terms of IC

(inhibitory

concentration). The concentration of antioxidant

compounds necessary to inhibit free radicals by 50%

is called the IC

value (Chow et al, 2003).

The efficacy of using herbal tamarind infused

water and drink is still largely based on empirical

data. Several studies related to the antioxidant activity

of tamarind and turmeric infused water and traditional

herbs are still limited, therefore this study aimed to

determine the proportion of tamarind turmeric IC

antioxidant in infused water and traditional herb

products, using the DPPH method.

2 MATERIALS AND METHOD

2.1 Materials

Turmeric from species Curcuma longa L. and

tamarind from species Tamarindus indica L.,

determination of turmeric is evaluated at the

Laboratory of Materia Medica Batu, Malang, and

tamarind at the Laboratory of Agrotechnology

University of Darussalam Gontor. The chemicals

used in this study include DPPH (1,1-diphenyl-2-

pikrihydrazil), ascorbic acid, methanol pro analysis,

aquadest, aluminum foil, and drinking water.

2.2 Preparation of Infused Water

Tamarind turmeric herbal traditional was made by

modification of Haitami (2017). Tamarind turmeric

infused water was made by cleaning the skin of the

turmeric rhizome, then washing it with running water.

Turmeric was weighed 25 g. While the tamarind fruit,

separated from the seeds, then the flesh is weighed as

much as 12.5 g. The two samples were put into a glass

bottle filled with 500ml of drinking quality water.

Infused water was allowed to stand for 12 hours at

room temperature. After 12 hours the turmeric acid-

infused water was concentrated using rotary

evaporator.

2.3 Preparation of Traditional Herbal

Tamarind turmeric herbal traditional was made by

modification of A’yunin (2019). Skin of the turmeric

rhizome was cleaning, then washing it with running

water. The rhizome was grated and weighed as much

as 25 g, while the tamarind fruit was separated from

the seeds and weighed as much as 12.5 g. After that,

grated turmeric rhizome and tamarind meat that has

been weighed and then boiled with 500 ml of boiling

water for 10 minutes. After 10 minutes, tamarind-

turmeric herbal solution was filtered through filter

paper and allowed to cool. After letting it cool down,

the tamarind turmeric herbal traditional was

concentrated by using rotary evaporator.

2.4 Preparation of Solution Test

2.4.1 Preparation of DPPH Stock Solution

DPPH was weighed 4 mg and dissolved in 100mL

methanol p.a. until concentration 40 ppm in a 100 ml

volumetric flask. The solution must be made fresh

and covered with aluminium foil.

2.4.2 Preparation of Ascorbic Acid

Standard Solution

Preparation of a standard ascorbic acid comparison

solution by dissolving 100 mg of ascorbic acid

powder into distilled water in a 100 ml volumetric

flask to obtain a solution with a concentration series

of 1000 ppm. Then pipette 1, 2, 3, 4, 5, 6, 7, 8, 9 ml

to obtain a solution with a concentration series of 100,

200, 300, 400, 500, 600, 700, 800, and 900 ppm.

2.4.3 Preparation of Tamarind- Turmeric

Solution Test

A total of 100 mg of the filtrate of the tamarind

turmeric herbal medicine was taken, then added

methanol to 100 ml, so that the concentration of the

test solution of the tamarind turmeric test solution

was 1000 ppm. Then pipette 1, 2, 3, 4, 5, 6, 7, 8, 9 ml

to obtain a solution with a concentration series of 100,

200, 300, 400, 500, 600, 700, 800, and 900 ppm.

Optimization of antioxidant activity test methods.

ICSDH 2021 - International Conference on Social Determinants of Health

228

2.5 Determination of the Operating

Time (OT)

A total of 1 ml of 400 ppm DPPH stock solution was

put into a 5 ml volumetric flask, then 3 ml of 400 ppm

sample was added. The solution is measured at the

maximum wavelength for ± 5-60 minutes.

Furthermore, the same treatment was carried out for

a solution of ascorbic acid.

2.6 Determination of the Maximum

Absorption Wavelength (λ

Maximum)

A total of 1 ml of 40 ppm DPPH stock solution was

added to 3 ml of the test solution in a 5 ml volumetric

flask. The solution was homogenized, allowed to

stand for OT, then scanned the maximum with a

visible spectrophotometer at a wavelength of 400-600

2.7 Antioxidant Activity Test

In a 10 ml volumetric flask, 2 ml of each stock

solution of DPPH was added, then 2 ml of the test

solution was added. The solution was added with

methanol, vortexed, and allowed to stand for OT. The

absorbance of the solution was measured at maximum

by visible spectrophotometry. The work was

replicated 3 times. Antioxidant activity is calculated

using the formula:

%𝐼𝐶





𝐴𝐵

𝐴

Note : A : Absorbance of DPPH solution

B : Absorbance of Sample solution

The data obtained from this study is by comparing

the absorbance value, which is the result of

antioxidant levels by calculating IC50 by using a

linear regression line equation between each

concentration of infused water and jamu turmeric acid

(x axis) with % IC (y axis). Then, statistical tests were

carried out using an unpaired T test for determined

the significance of the difference in IC50 values of

infused water and jamu turmeric acid. The IC50 data

of turmeric acid in the infused water and herbal

medicine preparations were then compared with the

IC50 of ascorbic acid. The use of ascorbic acid as a

comparison because ascorbic acid is one of the most

commonly used antioxidant compounds. In addition,

ascorbic acid belongs to the group very strong

antioxidant because ascorbic acid is one of the

antioxidant compounds that are pure without a

mixture of other compounds (Wibawa, 2020).

3 RESULTS AND DISCUSSION

3.1 Preparation of Turmeric-Tamarind

Infused Water and Traditional

Herb

The preparation of turmeric-tamarind traditional

herb, the part of the turmeric that was taken as the test

material in this study was the rhizome part. This

section was chosen because it was more yellow in

colour than the other parts, so it was believed to

contain more antioxidant compounds. The process of

washing the turmeric rhizomes should not be too long

because the water could carry the colour of the

rhizomes, which affected the antioxidant compounds

contained in the rhizomes. After washing, the

turmeric was grated and weighed, while the tamarind

part was extracted from the flesh. The tamarind was

separated from the seeds and then weighed. After

weighing the grated turmeric and tamarind pulp, the

mixture was boiled in boiling water for 10 minutes.

The herbs were cooled and then filtered.

In addition to being in the form of traditional

herbs, tamarind turmeric was made with infused

water. The difference between these two preparations

in the manufacturing process. The turmeric rhizome

which had been peeled and washed was weighed. The

same as turmeric tamarind, had been separated from

the seeds and then weighed. The weighed turmeric

rhizome and tamarind flesh were placed in a glass

bottle filled with 500 ml of drinking water. The

infused water was stored for 12 hours at room

temperature.

In this study, the samples to be analysed for

spectrophotometry were made in the form of filtering.

The method of using the filtrate was chosen to

facilitate the reading of absorbance by

spectrophotometry. Infused water and traditional herb

filtrate were prepared using a water bath, and the

filtrate performance was shown in the following

table:

Table 1: The result of concentrating tamarind turmeric in

infused water and traditional herb preparations.

Sample weight Total Yield

Preparation Filtrate

Turmeric-

tamarind 37,5 g

Infused water

183 g 20,2 g 11,03 %

Turmeric-

tamarind 37,5 g

Traditional

herb 194 g 55,8 g 28,76 %

The infused water filtrate was browner in colour,

while the herbal filtrate was more yellowish. This was

because, in the process of make tamarind-turmeric

Comparison of Antioxidant Activity between Turmeric- Tamarind Infused Water and Traditional Herbal using DPPH Method

229

traditional herb, the turmeric used was completely

crushed, while the turmeric-tamarind infused water

was only cut into small pieces and soaked for several

hours. Yield is the relationship between the number

of metabolites obtained after the extraction process

and the weight of the sample used. Based on percent

yield, traditional herb (28,76 %) are known to

perform better than infused water (11,03 %). Yield

data have something to do with the active compound

in a sample, so if the yield is higher, the number of

active compounds contained in the sample will also

increase (Dewatisari, 2017).

3.2 Determination of Maximum

Wavelength (λ Maximum)

The purpose of determined the maximum wavelength

in this study is to determine the wavelength at which

the DPPH solution has the maximum absorption. The

experimental conditions and the tools used were not

always the same, so the maximum wavelength

generated in each study might be different. If the

measurement was carried out at the maximum

wavelength, a small change in the concentration of

the solution to be analyzed could lead to a large

difference in the absorption results. This would

increase the sensitivity of the method (Yulia, 2015).

Figure 1: Result of the maximum λ determination curve.

The determination of the wavelength of maximum

absorption had been measured the wavelength of a

series of concentrations of DPPH, namely 40 ppm.

The maximum wavelength measurement was carried

out at a wavelength of 510 nm to 520 nm. This

wavelength range was chosen to see if there was a

change in wavelength compared to the theoretical

maximum wavelength, which was 517 nm. From the

determination of the wavelength carried out, the

maximum wavelength at 516 nm was obtained. This

wavelength shift is called a hypsochromic shift,

which is a shift in absorbance to a shorter wavelength

region due to the substitution or solvent effect

(Dachriyanus, 2004). Another possibility is a factor in

the difference in equipment and experimental

conditions.

3.3 Determination of Operating Time

(OT)

The operating time was the time that the absorption

reading was taken with a visible spectrophotometer,

where the test solution had perfectly reduced the

DPPH radical to obtain a stable absorbance value.

The operating time determination was performed by

measuring the relationship between absorbance and

measurement time.

Figure 2: The operating time of turmeric-tamarind

traditional herb and infused water. Blue line is for OT

traditional herbar and Red line is OT infused water.

In this study, the determination of OT for sample

of turmeric-tamarind traditional herb and OT

turmeric-tamarind infused water. The OT in this

study was measured from minute 5 to minute 40 at a

maximum wavelength of 516 nm. The OT

measurement results between the tamarind and

turmeric-infused water samples showed a time

difference, with a stable absorbance result of 0.355 at

15 minutes for turmeric OT traditional herb and a

stable absorbance result was 0.347 at 25 minutes for

OT turmeric-tamarind infused water. From the results

of the investigation, the most stable OT was selected

to minimize absorption variations and have a high

degree of reproducibility in the new measurement.

3.4 Results of the Determination of

Antioxidant Activity by the DPPH

Method

Measurement of the antioxidant activity of the

traditional herbs of tamarind and turmeric and the

water infused with tamarind was carried out at

various concentrations. This was done to determined

ICSDH 2021 - International Conference on Social Determinants of Health

230

the effect of concentration on antioxidant activity.

The higher concentration of sample solution, the

more hydrogen atoms were donated, so the

antioxidant activity was higher. DPPH was a stable

dark purple free radical that had a strong absorbance

at a wavelength of 517 nm (Molyneux, 2004). The

dark purple color of the DPPH solution was formed

because DPPH radicals could resonated to form long

chromophore groups.

Figure 3: The reaction of the radical DPPH with

antioxidants.

In this study, the DPPH solution was a solution

that acted as a control solution that had a higher

absorbance than the sample solution. This was

because the control solution only contained DPPH

and did not contain compounds that had antioxidant

activity. The turmeric-tamarind traditional herb and

infused water filtrate samples were diluted with

concentrations of 100, 200, 300, 400, 500, 600, 700,

800, 900 and 1000 ppm. Every 2 ml of sample

solution and 2 ml of 40 ppm DPPH solution were

added which were homogenized and allowed to stand

for OT for each preparation, then the absorbance was

measured used UV-Vis spectrophotometry. After

obtained the absorbance value, % inhibition

calculated using the formula:

%𝐼𝐶





𝐴𝐵

𝐴

After obtained the % inhibition, a linear

regression equation was performed to calculate the

antioxidant activity. Antioxidant activity was

determined based on the curve of the linear regression

equation between concentration and % IC. Therefore,

three repetitions were carried out to obtain a linear

regression equation. Linearity was expressed as the

correlation coefficient (r). The antioxidant activity

was expressed by IC

, which was the concentration

of antioxidants necessary to capture 50% of the

DPPH radicals. The lower the IC

value, the less

concentration was needed to scavenge free radicals.

According to the linear regression equation

between each concentration of tamarind-turmeric

traditional herb, water infused with tamarind

turmeric, and ascorbic acid with % IC, the IC

value

could be seen in the following curve:

Figure 4: IC

value of ascorbic acid, turmeric-tamarind

infused water, and traditional herb.

From the diagram above, it was known that

turmeric-tamarind traditional herb had an IC

value

of 94.21 ppm, and turmeric-tamarind infused water of

142.08 ppm. The smaller IC50 value, show the

greater the antioxidant activity. Both samples had

different groups of antioxidant activity according to

the IC

classification table below:

Table 2: Antioxidant power with DPPH reactive

compounds following Haerani, (2019).

Classification of IC

≤ 50 ppm Very strong

etween 50-100

m Stron

etween 101-150 ppm Moderate

more than 151 ppm Wea

From the table above, turmeric-tamarind

traditional herb was known to be a strong antioxidant,

and turmeric-tamarind infused water was included in

the moderate antioxidant group. This was probably

due to the difference in particle size between the two

preparations. Turmeric-tamarind traditional herb had

a smaller particle size because the ingredients used

were subjected to a refining process used a blender.

As for the infused water preparation, the ingredients

were only sliced with a knife. This was in line with

one of the extraction principles, that was the particle

size important to ensure that the extraction of the

compound could take place as much as possible. The

particle size was related to the area of the surface that

would be in contact with the solvent. (Sapri et al.,

2004). The main process in the transport of

compounds in infused water and traditional herb is

the diffusion process. Diffusion is the process of

moving a substance or particle from a highly

Comparison of Antioxidant Activity between Turmeric- Tamarind Infused Water and Traditional Herbal using DPPH Method

231

concentrated part to a low concentration part. Apart

from the particle size factor, another factor that

affects is the surface area of an area. The wider the

surface of a diffusion area, the wider the parts that can

be crossed.

In this study, the standard solution used was

ascorbic acid which had an IC

value of 5.8 ppm.

Ascorbic acid was used as the standard reference

solution because ascorbic acid is one of the most

common antioxidant compounds. Also, ascorbic acid

is included in the very strong antioxidant group

because ascorbic acid is one of the antioxidant

compounds that is pure without any other

compounds.

To determine the significant difference between

the IC

value of the turmeric-tamarind traditional

herb and the turmeric-tamarind infused water, a

statistical test was performed, namely the

independent T-test. The independent T-test requires

that the data be normally distributed. Therefore, the

first step to take was a normality test used the

Shapiro-Wilk test. This test had a sample requirement

of ≤ 50. The next step was to perform an analysis to

see the significance of the IC

value between

turmeric-tamarind infused water used an independent

T-test. The alternative hypothesis used was the IC

value of turmeric-tamarind traditional herb a was

different from turmeric-tamarind infused water. The

null hypothesis (H

) was that the IC

value of

turmeric-tamarind was not different from turmeric-

tamarind infused water. The results obtained with a

significance value of 0.000 between turmeric-

tamarind infused water. When compared to the

specified significance value, namely 0.05, H0 was

rejected because it was 0.000 < 0.05. Therefore, it

could be concluded that there was a significant

difference in IC

value between turmeric-tamarind

traditional herb and turmeric-tamarind infused water.

4 CONCLUSIONS

Turmeric-tamarind infused water (IC

142.08 ppm)

had lower antioxidant activity than turmeric-tamarind

traditional herb (IC

94.21 ppm). Based on statistical

tested, there were differences in the IC

value of

turmeric-tamarind in traditional herb and infused

water, influenced by differences in dosage forms and

the manufacturing process of these preparations.

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