Determination of Total Flavonoid Levels in Packaged Tea Bags

Combination of Dayak Onion and Beet Root with UV Visible

Spectrophotometric Method

Rahma Yulia

, Adewirli Putra

, Yulinda Rahmi

Mohammad Natsir University, Bukittinggi, Indonesia

Keywords: Total Flavonoid; Dayak Onion; Beet Root; Tea Bag; Antioxidants.

Abstract: The utilization of natural resources that contain antioxidants is in high demand by the community to prevent

degenerative diseases such as heart disease, diabetes, stroke and cancer. Processing of Dayak onion and beet

root in the form of herbal tea has great potential to be used as a functional drink because both of these plants

contain flavonoids which have antioxidant properties. In 5 tea formulas made by comparing the composition

of Dayak onions and beet root, a preference test was conducted on several panelists related to color, aroma

and taste and obtained 3 formulas favored by the panelists are formula1, 3 and 4. the purpose of this study

was to determine the total flavonoid levels of each of the teabag formulas favored by panelists using the UV

Visible spectroprotometry method with a quercetin comparison. From the research conducted obtained linear

regression equation data y = 0.0364x + 0.0028 with r = 0.997. Total flavonoid levels for each teabag

combination of dayak onion and beet root for formula 1; formula 3; formula 4 is 0.9309%; 0.975% and

1.841%, where the highest total flavonoid content is formula 4, which is 1, 841%.

1 INTRODUCTION

Bukittinggi is one of the tourism city in West

Sumatra, which is famous for its various culinary

delights and delicious flavors. Many culinary

preparations using spices and coconut milk as the

basic ingredients. The pattern of life of the people of

Bukittinggi who often consume foods containing

coconut milk and fatty foods causes the people of

Bukittinggi to have great potential to get a

degenerative diseases such as heart disease, diabetes

and stroke. The high price of modern medicines

encourages consumers to try other alternatives by

using the trend back to nature to maintain their health.

one of the natural product that have the potential to

overcome these degenerative diseases is plants that

contain antioxidant compounds.

Utilization of natural resources that contain

antioxidants is in high demand by the community to

prevent degenerative diseases. Antioxidants are

substances that at low concentrations can prevent or

slow down the oxidation process by binding to free

radicals and highly reactive molecules so that cell

damage can be inhibited. This compound has a small

molecular weight but is able to inactivate the

development of oxidation reactions by preventing the

formation of radicals (Dimitrios, 2006). Antioxidants

are generally present naturally in an important role for

the protection of body health. This compound can

prevent oxidative damage and reduce the risk of

disease.

Among the plants that contain lots of antioxidants

are Dayak onions (Eleutherine Palmifolia) and

beetroot (Beta vulgaris). Both of these plants have

identical colors that are purplish red which contain

bioactive compounds such as phenols, flavonoids,

taanin, glycosides, steroids and alkaloids (Claudea,

2013, Puspadewi, 2013). purplish red color in both

plants is caused by anthocyanin pigment content.

Anthocyanins are a group of pigments that cause

reddish color, located in water soluble cells in water

(Jaya 2013). Besides functioning as an antioxidant,

anthocyanin has other uses including as a natural

indicator, and as a coloring agent textile and food

industries (Hendrawan 2011). Several studies have

been carried out using anthocyanin pigments from

beetroot and dayak onions for natural dyes in food or

natural dyes for cosmetics such as lipsticks, but no

studies have been reported regarding the manufacture

of functional drinks from these two plants.

Diversification of natural products that are

traditionally processed can be done one of them by

356

Yulia, R., Putra, A. and Rahmi, Y.

Determination of Total Flavonoid Levels in Packaged Tea Bags Combination of Dayak Onion and Beet Root with UV Visible Spectrophotometric Method.

DOI: 10.5220/0009515103560363

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 356-363

ISBN: 978-989-758-460-2

making herbal tea drinks that are packaged in dyed

containers like tea drinks in general. Herbal Tea is

one of the tea beverage products from herbal plants

that has properties in helping the treatment of an

illness or as a body refreshing drink (Hambali et al,

2005, Mun’in, 2008). This product is a form of

change in health products.

Processing of Dayak onions and beetroot in the

form of herbal tea has great potential to become a

functional beverage, because it is related to its natural

antioxidant content, both plants can be utilized to

protect the body from free radical attack which results

in degenerative diseases.

Research conducted by Yulia et al, 2019,

regarding the formulation of tea bags with a

combination of dayak onions and beetroot with five

formulas containing different composition of dayak

onions and beetroot. After the hedonic test related to

the color, smell, taste and shape of the 5 formulas and

obtained three formulas favored by the panelists

namely formulas 3, 1 and 4.

Three formulas are preferably carried out

quantitative analysis of total flavonoid levels to find

out how much flavonoid levels are measured in the

tea packaging combination of dayak onions and

beetroot. So that it can be chosen among the three

formulas that have the highest flavonoid content so

that it can be developed into a health functional

beverage product. Determination of total flavonoid

levels was carried out by the method of chang et al

2002 using UV Visible Spectrophotometry.

2 MANUSCRIPT PREPARATION

2.1 Material

The Material used include five packaged teabag

formulas that have been studied by Yulia, et. al

(2019), Ethanol 95%, Methanol, Pure Quercetin,

Aquadest, 10% AlCl3 buffer solution, Sodium

Acetate buffer solution, Ethyl acetate, 4N HCl

solution.

2.2 Tool

The tools used in this study include a funnel, beaker

glass (pyrex), erlenmeyer (pyrex), stir bar, measuring

flask (pyrex), volume pipette (pyrex), test tube,

measuring cup (pyrex), UV-Vis spectrophotometer

1800 (pyrex) Shimadzu), pH meter.

2.3 Research Stages

There are three stages of research in this study the first

is the preparation and formulation of tea bags (Yulia,

et.al. 2019). The second stage was the packaging of

tea bags using 95% ethanol and the preparation of

standardized quercetin solutions and test solutions,

and the third step was the determination of total

Flavonoid and Anthocyanin levels using a UV-Vis

spectrophotometer.

2.3.1 Formula Teabag Combination of Beet

root-Dayak Onion

There are five Dayak onion-beetroot teabag formulas

made by comparison of different combination

ingredients (Yulia et al. 2019) as shown in Table 1.

Table 1. Beetroot - Dayak Onions Teabag Formulas

Formula

Code

Beet

Root

(gram)

Dayak

Onion

(gram)

Stevia

Leaves

(gram)

1,5

0,5

0,15

0,75

1,25

0,15

0,5

1,5

0,15

0,25

1,75

0,15

2.3.2 Manufacture of Extractions, Standard

Solutions and Test Solutions

Extraction

Weigh each sample of tea bags as much as 5 grams.

Enter each sample that has been weighed into each

100 ml measuring flask. Add 100 ml of 95% ethanol,

cover. Figure 1 is Maceration for 24 hours. The first

6 hours shake it many times. And leave it for 18

hours. Then filter the results of maceration.

Figure 1: maceration process of sample.

Quercetin Standard Solution

Pure quercetin weighed as much as 25 mg, dissolve

with methanol in a 100 ml volumetric flask to mark

the mark. This result is used as a standard solution.

The standard solution is then diluted with methanol in

6 different concentrations (Figure 2). Each

concentration was pipetted 2 ml, then 0.1 ml AlCl

Determination of Total Flavonoid Levels in Packaged Tea Bags Combination of Dayak Onion and Beet Root with UV Visible

Spectrophotometric Method

357

10% reagent was added, 0.1 ml sodium acetate, and

2.8 ml Aquadest, homogeneous and incubated for 30

minutes at room temperature. The absorbance was

measured on a UV-Vis 415 nm spectrophotometer

using blank solution without quercetin and AlCl

Figure 2. Quercetin Standard Solution

Sample Solutions

The extract that was made was weighed as much as 1

gram, then hydrolyzed with 4 ml HCl as much as 2 ml

for 30 minutes in a measuring flask. The solution is

then filtered and concentrated. Then the extract was

filtered with 15 ml of ethyl acetate, 3 times, the ethyl

acetate fraction was collected and concentrated. The

results of the ethyl acetate extract were put in a 25 ml

measuring flask and dissolved with methanol to the

limit mark (test solution). Do the same for the other 2

samples.

2.3.3 Determination of Total Flavonoid

Levels and Determination of

Anthocyanin Levels in Five Formulas

Was Carried out by the Chang Method

Determination of Total Flavonoid Levels

The test solution was pipetted as much as 0.5 ml, then

dissolved with 1.5 ml of methanol in the test tube,

then add a reagent consisting of 0.1 ml of 10% AlCl

0.1 ml of Sodium Acetate and 2.8 ml of distilled

water, homogeneous and incubation for 30 minutes at

room temperature. The absorption solution was

measured on a UV-Vis 415 nm spectrophotometer

using blank solution without the addition of AlCl

replaced with distilled water. Measurements were

made three times, levels were calculated as averages.

The total flavonoid content is expressed by

comparison equivalents of quercetin. Do the same

thing in the other 4 samples.

Determination of Anthocyanin Levels of Dayak

Onions – Beet root teabag

Determination of anthocyanin levels was done by

UV-Vis Spectrophotometer. The test solution was

made 5 grams of a mixture of tea combination of

dayak onions and beetroot extracted with 250 ml of

water for 5 minutes and 10 minutes. The test solution

was taken 1 ml, then two samples were measured

against 5 ml of pH solution 1.0 (buffer solution of

Potassium Chloride) and 4.5 (buffer solution of

Sodium Acetate). Furthermore, it was analyzed with

a UV-Vis spectrophotometer at a wavelength of 520-

700 nm.

3 RESULTS AND DISCUSSION

Beetroot is the taproot portion of a beet

plant, usually known in North America as the beet,

and also known as the table beet, garden beet, sugar

beet, red beet, dinner beet or golden beet. It is one of

several cultivated varieties of Beta vulgaris grown

for their edible taproots and leaves (called beet

greens), they have been classified as B.

vulgaris subsp. vulgaris 'Conditiva' Group. Besides

being used as a food, beets have uses as a food

colouring and as a medicinal plant. Many beet

products are made from other Beta vulgaris varieties,

particularly sugar beet.

In preliminary research, beetroot juice

reduced blood pressure in hypertensive

people. Tentative evidence has found that dietary

nitrate supplementation, such as from beets and other

vegetables, results in a small improvement in

endurance exercise performance. The red colour

compound betanin is not broken down in the body,

and in higher concentrations may temporarily cause

urine or stools to assume a reddish colour, in the case

of urine a condition called beeturia.

Although

harmless, this effect may cause initial concern due to

the visual similarity to what appears to be blood in the

stool, hematochezia (blood passing through the anus,

usually in or with stool) or hematuria (blood in the

urine). Nitrosamine formation in beet juice can

reliably be prevented by adding ascorbic acid.

Dayak onions are small bulbs that produce

several long, ribbon-like leaf stalks with a single

flowering stem. The smooth, dark red bulbs are

shaped like rounded diamonds with small, wispy

brown roots extending from the ends.

Research on Dayak onions has been carried out,

including plant bulbs of the genus Eleutherine

(Eleutherine bulbosa and Eleutherine Americana),

which are known to contain secondary metabolites of

the naphthoquinone group (elecanacin, eleutherin,

eleuthero, eleuthero). Onion Dayak has anti cancer

and anti oxidants, which are usually found in vacuole

cells in the form of glycosides. Some studies also

state the content of the active compounds in Dayak

onions is extensive, so it is very reasonable for

various properties. These compounds include

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

358

alkaloids, steroids, glycosides, flavonoids, phenolics,

tannins, and saponins. One of these compounds,

namely flavonoids, can be efficient as anticancer,

antiviral, anti inflammatory, reduce the risk of

cardiovascular disease, and free radical catchers.

According to D. Lestari et all, 2018, dayak

onion/tiwai onion contain secondary metabolites such

as alkaloids, flavonoids, tannins, and quinones. These

compounds are known to have a very broad biological

activity including antioxidants and anticancer.

Flavonoids are known to be good antioxidants

because they have at least two hydroxyl groups in

ortho positions and para which can capture free

radicals by freeing hydrogen atoms from their

hydroxyl groups. Flavonoids as antioxidants have a

higher potential as anticancer drugs than vitamins and

minerals. Flavonoid compounds can prevent the

reaction of carcinogen molecules joining cell DNA so

as to prevent cell DNA damage; the bioactive

components of flavonoids can prevent the initial

process of cancer cell formation. Flavonoids can

stimulate the regenaration process of mutated DNA

cell so that the cells become normal again. In

addition, quinone compounds have also been reported

to have antioxidant activity. It is likely that quinone

originates from the oxidation of the corresponding

phenols namely catechol to form ortho-quinones and

quinol producing para-quinones.

Beetroot and dayak onions in this research

obtained from palano stone plots in Agam Regency,

West Sumatra Province can be seen in Figures 1 and

2. Beetroot are used which have ± 10 weeks old.

Dayak onions used are ± 4 month old, onions or plants

have flowered, this is because the quality of the

Dayak onions is in an optimal state. Simplicia is

cleaned with running water to remove impurities

(Puspadewi, 2013). After the simplicia is selected

and cleaned, then thinly sliced to be dried using an

oven at 45 ± 2oC until perfect drying is obtained,

because the simplicia cannot be heated at high

temperatures because it will cause damage or loss of

secondary metabolites. This drying process intended

to reduce water content contained in the sample, so it

can prevent spoilage by bacteria. After the dried

simplicia is then made into powder using a blender,

the powder is weighed for each formula and packaged

into a tea bag.

Figure 3: Dayak Onion Figure 4: Beetroot

The packaging of tea bags is carried out with five

different formulations namely F1, F2, F3, F4 and F5

formulas. The results of the tea bag can be seen in

Figure 5.

Figure 5: Forms of Steeping five combination teabag

formula (Yulia,et.al.2019).

This Dayak onion-beetroot combination teabag is

one of the more practical forms of preparation,

preferred by the public and has antioxidant properties,

because each of the simplicia that has been previously

studied (Melisa, 2015 and Puspadewi, 2013) has a

compound content. Compounds that act as

antioxidants in simplicia include Flavonoids and

Anthocyanins. To determine the content of these

compounds in this teabag, the flavonoid content was

tested by UV-Vis spectrophotometer by Chang

(2002). Before conducting the test for determining the

level of flavonids carried out on this teabag extract

based on the Chang method. The extraction process is

aimed at take chemical compounds contained in the

sample. The principle of extraction is based on

displacement the mass of the components of the

substance dissolved into the solvent resulting in

displacement in the interface layer and diffuses into

the solvent (Harborne, J.B 1987).

To get chemical compounds that are the desired

extraction method is used is a method of extracting

nutritious substances or substances active parts of the

plant by using solvents appropriate (Yuliani &

Satuhu, 2012). The extraction method used on this

research is maceration, because of this method

simpler, easier and without heating. Because heating

can make flavonoid levels reduced. The calibration

curve with a standard solution of Quercetin which is

made with 6 concentrations shown in Table 2.

Determination of Total Flavonoid Levels in Packaged Tea Bags Combination of Dayak Onion and Beet Root with UV Visible

Spectrophotometric Method

359

Table 2. Concentrations of quercetin standard solutions on

calibration curves

Concentrations

(ppm)

Absorbant (A)

0,215

0,360

0,533

0,659

0,792

Quersetin used (Figure 6) as a standard solution

because quercetin is flavonoid flavonol group which

has a group keto at C-4 and has a hydroxyl group on

neighboring C-3 or C-5 atoms from flavones and

flavonols (Azizah and Faramayuda 2014)

Figure 6: Quercetin calibration curve

The calibration curve obtained is y = 0.0364x +

0.0028, with r = 0.997. A value of r close to 1

indicates a linear calibration curve and there is a

relationship between the concentration of quercetin

solution and the absorption value, Azizah (2014).

This method was chosen because it is easier and

simpler, faster, economical, and is known to be more

specific to the flavonoid and flavonol groups.

Aluminum (III) chloride reagents are used to form

acid-resistant complexes with C-4 ketone groups and

C-3 or C-5 hydroxyl groups in flavones and flavonols,

and form acid-resistant complexes with ortho-

hydroxy groups in the ring A or B in flavonoids

(Chang et al., 2002 & Humadi,Istudor,2008). In the

measurement of total flavonoid compounds, the

sample solution is added AlCl

which can form

complexes, resulting in a shift in wavelength towards

the visible which is indicated by the solution

producing a more yellow color. And the addition of

potassium acetate which aims to maintain

wavelengths in visible areas (Chang et al, 2002). The

addition of aluminum chloride aims to form

complexes with quercetin (Indrayani, 2008). The

incubation treatment for 1 hour before the

measurement is intended so that the reaction runs

perfectly, so that the resulting color intensity is more

maximal (Azizah and Faramayuda 2014). Total

flavonoid levels are calculated as equivalence of

quercetin raw materials (Humadi, Istudor, 2008).

In general, the determination of total flavonoid

levels in a plant sample is based on the formation of

aluminum complex compounds (Al-Flavonoids), in

the form of a yellow solution. The addition of acetate

salts to the determination of flavonoid levels is

intended to produce shifts and stronger peak

absorbance intensities (Pekal & Pyrzynska, 2014).

Al-Flavonoid chelate complexes are formed in ketone

groups and hydroxyl groups of flavonoids

(Sepahpour, Selamat, Manap, & Razis, 2018), the

reactions that occur can be seen in Figure 7.

Figure 7: Chemical reaction of Flavonoid with AlCl

From this method, the results of determining the total

Flavonoid levels are obtained in Table 3.

Table 3: Determination of total flavonoid levels of teabag

combination with beetroot and dayak onions.

Extract

weight

Absorbant

at λ 436,5

nm (y)

Total

flavonoid

levels (%)

Average total

flavonoid levels

(%)

0,175 g

2.372

0,9309

1,249

0,146 g

2.072

0,975

0,105 g

2,815

1,841

the range of total flavonoid levels = 0.9307-1.841%

Total flavonoid levels obtained for formula 1

teabag combination of beetroot and dayak onions is

0.9303%, for formula 3 is 0, 975% and total flavonoid

level for formula 4 is 1.841% with an average total

flavonoid level is 1, 249%

Determination of anthocyanin levels was carried

out on the three best formulas from the results of the

study (Yulia, et.al.2019). Determination of the levels

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

360

(Figure 8) of anthocyanin extracted in the same way

according to the way the tea is served, which is

brewed for a few minutes in accordance with the way

tea is served in general, in the experiment 5 minutes

and 10 minutes are chosen to see the levels of

anthocyanin with different extraction times. Figure 8

explains the sample solution for determining

anthocyanin levels.

Figure 8: Sample solution for determination anthocyanin

levels.

Anthocyanins are included in the class flavonoid

compounds, constitute a group the biggest natural

pigment in plants which dissolve in ak which is

responsible for give color to flowers, fruit and

veegetables. Anthocyanins can be beneficial for

health as a source of antioxidants. This is due to the

compound This polyphenolic is a derived glycoside

polyhydroxy and polymethoxy from, 2-

phenilbenzopirilium or flavilium salt.

Anthocyanins are in several equilibrium form.

Kinbtika study and thermodynamics that are studied

in general accepting the transformation of differences

(proton transfer, isomerization and tautomerization)

flavilium cation from simple anthocyanin under

various pH conditions. In acidic solution stronger

(under pH 2) more flavilium cations dominant and

provides anthocyanin solutions the Red one.

The anthocyanin stability is not only influenced

by the heating temperature in the processing only, but

also influenced by intrinsic factors and extrinsic in the

product, such as pH, temperature storage, chemical

structure and concentration existing anthocyanins, the

presence of light, oxygen, enzymes, proteins, and

metal ions. to know the stability of anthocyanin is

needed initial data on anthocyanin levels from the

starting material contain these substances.

Determination of anthocyanin is done with the pH

difference method of pH 1.0 and pH 4.5. At pH 1.0

anthocyanin is formed oxonium compound. The

increasingly situation especially if it is nearing pH I

will cause more pigment anthocyanins are in the form

of cations colored flavilium or oxonium and

absorbance measurement will show an increasingly

large amount of anthocyanin. At pH 4.5, it is on weak

acids Flavilium cations change into more forms stable

colorless hemiketal and shape calchont (Figure 9).

Orange-purple flavilium cation colorless hemiketal for

pH 1.0 pH 4.5

Figure 9: Flavilium cation structure and hemiketal

shape R=H Glicocyde substitutens.

Measurement of anthocyanin levels using UV-

Vis Spectrophotometry, as previous studies

anthocyanin levels were calculated using the general

equation :

Concentracion (mg/ml) = A x MW x FD x 1000

/ (ϵ x l)

Information :

A = absorbance

= A 535.2 (pH 1.0) - A535.2 (pH 4.5)

MW = molecular weight = 433.2 g / mol

FD = dilution factor

ϵ = molar absorption = 31600 L / cm mol

l = cuvette width (1 cm)

Determination of anthocyanin levels was carried

out three times (Triplo) at the maximum wavelength

obtained which is 518 nm, the maximum wavelength

is then used to measure the absorption of the

calibration curve and extract sample (Azizah, 2014).

It is known that the main wavelength of anthocyanin

is in the range of 475-560 nm (Harborne, 1987). From

the results of determining the level of anthocyanin

obtained it can be said that the brewing of tea for 5

minutes and 10 minutes is almost the same so that the

brewing time can be recommended for only 5

minutes. Anthocyanin levels were determined using

UV-Vis spectrophotometry, based on the ability of

anthocyanins to produce colors at pH 1.0 (buffered

potassium chloride solution) and pH 4.5 (sodium

acetate buffer solution). The measurement data using

UV-Vis spectrophotometer is shown in Table 4.

Determination of Total Flavonoid Levels in Packaged Tea Bags Combination of Dayak Onion and Beet Root with UV Visible

Spectrophotometric Method

361

Table 4. Determination of anthocyanin levels of teabag

extract combined with beetroot and dayak onions

This characteristic depends on the transformation

of the chromophore structure. The color of the

anthocyanin ion stands out at pH 1.0. While the

colorless hemiketal structure at pH 4.5 (Herrera,

2004)

4 CONCLUSIONS

Determination of total flavonoid and anthocyanin

levels. The results obtained for total flavonoid levels

were tested on the three best formulas obtained in

previous studies (Yulia, et.al. 2019), namely the F1,

F3 and F4 formulas respectively were obtained

0.9309%, 0.975%, 1.841%, Whereas for anthocyanin

levels during 5 minutes, 5,849 mg / ml, 3,199 mg /

ml, 2,405 mg / ml were obtained. And for 10 minutes,

6.826 mg / ml, 4.535 mg / ml, 2.736 mg / ml were

obtained.

ACKNOWLEDGEMENTS

Acknowledgement to the Ministry of Research and

Technology of Higher Education for funding this

study through the 2019 Beginner Lecturer Research

grant.

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Spectrophotometric Method

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