Enhancement of Flavonoid and Anthocyanin Levels of Herbal

Beverage Containing Butterfly Pea Flower and Lemon Fruit to

Improve Anti-oxidant Activity

Fista Utami

, Dono Indarto

and Shanti Listyawati

Postgraduate Program of Nutrition Science, Universitas Sebelas Maret, Surakarta, Indonesia

Department of Physiology and Biomedical Laboratory, Faculty of Medicine, Universitas Sebelas Maret,

Surakarta, Indonesia

Biology Department, Faculty of Mathematic and Science, Universitas Sebelas Maret, Surakarta, Indonesia

Keywords: Herbal Beverage, Flavonoid, Anthocyanin, Butterfly Pea Flower, Lemon Fruit.

Abstract: Butterfly pea flower (Clitoria ternatea L.) and lemon (Citrus limon) have bioactive compounds such as

flavonoids and anthocyanin that were known the potential to be processed as functional beverage for health.

The combination of them was never been done before. This experiment aimed to analyze the levels of total

flavonoids, anthocyanin, and antioxidant activity in butterfly pea flower and lemon beverage. This

experimental research was experimental method while dried butterfly pea flower was brewed at temperature

of 60 degrees Celsius for 15 minutes combined with lemon fruits with formulation TL (100:0%) F1 (75:25%),

F2 (50:50%), F3 (25:75%), and JL (0:100%). The formulation beverages were analyzed for total flavonoid

by spectrophotometric method, anthocyanin by pH differential method, and antioxidant activity IC50 by

DPPH method. The formulation beverage of butterfly pea flower and lemon F3 (25:75%) was the best formula

based on the results for total flavonoids and antioxidant activity. Besides, formula F1 (75:25%) was the best

in anthocyanin levels. More composition butterfly pea flower in the beverage, contained more the flavonoid

and anthocyanin. Adding more lemon fruits into beverage made greater antioxidant activity IC50. Further

research is needed to examine other phytochemical compounds and the effect of its administration on health.

1 INTRODUCTION

Butterfly pea flower and lemon are quite commonly

found in Indonesia. The butterfly pea flower is a

flower from the Fabaceae family with the

international name blue pea flower because it is blue

which indicates the presence of anthocyanin

(Lestario, 2017). In addition, lemon is one of the

fruits from the genus Rutaceae which has the highest

hesperidin content compared to other citrus types

(Chaturvedi et al., 2016; Klimek-szczykutowicz et

al., 2020). No one has ever studied the total content

of flavonoids and anthocyanin related to the

combination of them.

Anthocyanin are natural pigments from fruits,

vegetables, and edible flowers. Anthocyanin are

glycosides (3-O-β-glucosidase) that release

anthocyanidin aglycones through glycosidases with 6

types of aglycones, including; cyanidin, delfinidine,

pelargonidine, malvidin, peonidin, and petudinine. In

addition, anthocyanin have the effect of inhibiting

DNA fragmentation by H

, inhibiting LDL-

induced Cu

oxidation and improving mitochondrial

function (Sunarti, 2021)

Delfinidine are known to reduce triglyceride

accumulation, adipogenesis and lipogenesis by

activating Adenosine Monophosphate-Activated

Protein Kinase (AMPK) and decreasing the

expression of Peroxisome Proliferator Activator

Gamma-Receptor (PPAR-γ). AMPK activation

inhibits the Acetyl Co-A Carboxylase (ACC) enzyme

in fatty acid synthesis and the HMG-CoA reductase

enzyme in cholesterol synthesis, and increases

adipose triglyceride lipase (ATGL) in fatty acid

catabolism resulting in lipolysis (Miey Park, 2019;

Parra-Vargas et al., 2018). This causes the butterfly

pea flower to potentially lower lipid profile levels and

reduce fat accumulation.

Furthermore, flavonoids of the flavanone group

such as hesperidin and its aglycone namely hesperetin

have several biological activities, including; as an

Utami, F., Indarto, D. and Listyawati, S.

Enhancement of Flavonoid and Anthocyanin Levels of Herbal Beverage Containing Butterﬂy Pea Flower and Lemon Fruit to Improve Anti-oxidant Activity.

DOI: 10.5220/0011642600003608

In Proceedings of the 4th International Conference on Social Determinants of Health (ICSDH 2022), pages 53-59

ISBN: 978-989-758-621-7; ISSN: 2975-8297

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

antioxidant, anti-inflammatory, and anti-carcinogenic

vitamin. Hesperidin upregulates the expression of

Nuclear Factor Erythroid-2 related factor 2 (Nrf2) and

Extracellular Signal Regulated Kinases 1 and 2 (ERK

1/2) genes which stimulate upregulation of Heme

Oxygenase-1 (HO-1) expression, resulting in a

decrease in intracellular prooxidants. In addition to

HO-1 expression, the Nrf2 gene also increased levels

of antioxidant enzymes such as glutathione

peroxidase, superoxide dismutase, and catalase.

Hesperidin also inhibits the formation of Advanced

Glycation End Products (AGEs) which triggers the

accumulation of extracellular protein damage so as to

prevent premature aging and degenerative diseases

(Sunarti, 2021).

Hesperidin is also known to suppress appetite by

stimulating the release of the appetite-regulating

hormone cholecystokinin into enteroendocrine cells,

decreasing the expression of LDL receptor coding

genes, increasing AMPK expression which plays a

role in increasing the oxidation of glucose and fat to

become energy, and inhibiting lipid metabolism

pathways on expression by PPAR-γ (Xiong et al.,

2019). This causes lemons to have the potential to

reduce lipid profile levels, body weight, and

premature aging.

Butterfly pea flower and lemon are available in

Indonesia in sufficient quantities to be developed.

This experiment aimed to analyze the levels of total

flavonoids, anthocyanin, and antioxidant activity in

combination beverage of butterfly pea flower and

lemon. The beverage is expected to be developed into

a functional beverage for the prevention and

treatment of diseases such as obesity, dyslipidaemia,

and premature aging.

2 MATERIALS AND METHODS

This experimental study was carried out at the Central

Laboratory of Food and Nutrition Studies, Gadjah

Mada University Yogyakarta and Food Technology

Laboratory, Faculty of Agriculture, Universitas

Sebelas Maret, Surakarta from January to February

2022. Dried butterfly pea flowers were obtained from

a restaurant garden, Pringsewu, Lampung Province,

Indonesia. Lemon fruits were obtained from a local

lemon plantation at Batu City, East Java Province,

Indonesia.

2.1 The Preparation of Butterfly Pea

Flower and Lemon Beverage

The preparation of the beverage referred to a previous

study Purwaniati et al., (2020) with slightly

modification. In brief, fresh butterfly pea flowers

were picked up from a plantation and then were dried

under sunlight during the day for 4-5 days. Dried

butterfly pea flowers were brewed using boiled

mineral water (60

C) 1:100 in ratio for 15 minutes

and lemon fruits were freshly harvested in 1-2 weeks

before the research began. The lemon fruits were

squeezed and which directly mixed with dried

butterfly pea flower brew. A combination beverage of

butterfly pea flower and lemon fruits was categorized

into three groups: F1 (75:25%), F2 (50:50%), F3

(25:75%) respectively (Table 1).

Table 1: The formulation of butterfly pea flower and lemon

beverages.

Materials

Formulation

TL F1 F2 F3 JL

Butterfly

ea flower (ml)

100 75 50 25 0

Lemon fruits

(

)

0 25 50 75 100

Total

(

)

100 100 100 100 100

2.2 Total Flavonoid Measurement

Determination of total flavonoid levels using UV-Vis

spectrophotometry method and was carried out by

weighing 1-2 g of the beverage sample and then

dissolving it in 10 ml of 96% ethanol. The solution

was then taken 1 ml of the solution and added 5 ml of

FeCl

solution to form a red colour. The solution was

added with 96% ethanol so that the final volume

became 10 ml. The absorbance was read with a

spectrophotometer at a wavelength of 520 nm

(Martini et al., 2020).

2.3 Anthocyanin Measurement

Determination of total anthocyanin levels using pH

differential method and was carried out by diluting a

combination beverage of butterfly pea flower and

lemon with KCl buffer pH 1 until an absorbance was

found to be less than 1.2 with a wavelength of 510

nm. After that, two types of samples were prepared,

namely the first sample using KCl buffer at pH 1 and

the second sample using Na-acetate buffer with pH

4.5. Both samples were read at a wavelength of 510–

750 nm (Pasaribu et al., 2021).

ICSDH 2022 - The International Conference on Social Determinants of Health

2.4 Antioxidant Activity Measurement

Determination of an antioxidant activity using

Diphenyl Picryl Hydrazyl (DPPH) method and was

carried out by weighing the sample 1 - 2 g, dissolving

with methanol at a certain concentration. After that, 1

ml of base liquor was taken and put into a test tube

and added 1 ml of 1,200 micro molar DPPH solution

and incubated in a dark room for 30 minutes. After

that, making solution contained 1 ml of DPPH

solution and 4 ml of ethanol. The solution was

calibrated at a wavelength of 515 nm and the total

antioxidant capacity was obtained.

Antioxidant Capacity.= OD blank–OD sample x 100%

OD blank

After obtaining the percentage of antioxidant

capacity, then a graph is made between the

concentrations of with the average % antioxidant

activity so that the value of y = bx + a is obtained,

then calculated IC50 value (Antarti & Lisnasari,

2018).

2.5 Statistical Analytic

The data were tested using One Way Anova Post Hoc

Tukey HSD to analyze the difference result and the

greatest of total flavonoids, anthocyanin, and

antioxidant activity IC50 in the beverages.

3 RESULTS

3.1 Total Flavonoids Levels

From Table 2, it indicated that total flavonoids levels

increased in combination of butterfly pea flower and

lemon beverages regarding to increased percentage of

lemon fruits. The highest total flavonoids was found

in the F3 (1,391.5±72.83 ppm), significantly different

from other formulation (p<0.001). The F2 and F3

formulas had significantly higher of total flavonoids

levels than the TL formula (Table 3).

Table 2: Total Flavonoids of Combination between

Butterfly Pea Flower and Lemon Beverages.

Formulation Total Flavonoid (ppm)

TL (100%) 881.00±24.04

JL (100%) 756.50±12.02

F1 (75:25%) 993.50±6.36

F2 (50:50%) 1,146.5±81.31

F3 (25:75%) 1,391.5±72.83

P value 0,001*

Note: *significant

In contrast, significant higher of total flavonoids

levels were observed in the F1-F3 formulas,

compared to the JL formula (p=0.003; 0.027; <0,001,

respectively). Besides, F3 formula was significant

higher of total flavonoids levels compared to the F1

and F2 formulas (p=0.003; 0.023, respectively).

Table 3: Mean Differences of Total Flavonoids in

Combination Between Butterfly Pea Flower and Lemon

Beverages.

Formulation

Differences

Mean Difference

(ppm)

P Value

TL (100%)

(

75:25%

)

-112.50 0.300

(

50:50%

)

-265.50 0.017*

(25:75%)

-510.50 0.001*

JL (100%)

(

75:25%

)

-237.00 0.027*

(

50:50%

)

-390.00 0.003*

(25:75%)

-635.00 <0.001*

TL (100%)

(100%)

124.50 0.234

(75:25%)

(

50:50%

)

-153.00 0.130

(25:75%)

-398.00 0.003*

(50:50%)

(25:75%)

-245.00 0.023*

Note: *significant

3.2 Anthocyanin Levels

Table 4: Anthocyanin Levels of Combination between

Butterfly Pea Flower and Lemon Beverages.

Formulation Anthocyanin (ppm)

TL (100%) 592.50±0.45

F1 (75:25%) 424.41±25.75

F2 (50:50%) 205.42±15.58

F3 (25:75%) 68.44±1.56

P value 0,001*

Note: *significant.

Anthocyanin levels in the beverage formulas reduced

in conjuction of decreased percentage of butterfly pea

flower concentration (Table 4). Table 5 showed that

the highest anthocyanin level was found in the TL

formula (592.50±0.45 ppm), which significantly

differed from the F1 (424.41±25.75 ppm, p=<0.001),

F2 (205.42±15.58 ppm, p=<0.001) and F3

(68.44±1.56 ppm, p=<0.001). F1 formula had the

Enhancement of Flavonoid and Anthocyanin Levels of Herbal Beverage Containing Butterﬂy Pea Flower and Lemon Fruit to Improve

Anti-oxidant Activity

highest anthocyanin levels compared to F2 and F3

formulas with very significantly different.

Table 5: Mean Difference of Anthocyanin Levels in

Combination Between Butterfly Pea Flower and Lemon

Beverages.

Formulation Differences

Mean

Difference

(ppm)

P Value

(100%)

F1 (75:25%)

168.09 <0.001*

F2 (50:50%)

387.08 <0.001*

F3 (25:75%)

524.06 <0.001*

(75:25%)

F2 (50:50%)

218.99 <0.001*

F3 (25:75%)

355.97 <0.001*

Note: *significant

3.3 Antioxidant Activity

Table 6: Antioxidant Activity of Combination Between

Butterfly Pea Flower and Lemon Beverages.

Formulation Antioxidant Activity

IC50 (ppm)

F1 (75:25%) 11,287.49±123.70

F2 (50:50%) 8,267.80±130.63

F3 (25:75%) 7,594.55±172.80

P value 0,001*

Note: *significant

Table 7: Mean Differences in Combination Between

Butterfly Pea Flower and Lemon Beverages.

Formulation

Differences

Mean

Difference(ppm)

P Value

(75:25%)

(50:50%)

3,019.69 <0,001*

(25:75%)

3,698.44 <0,001*

(50:50%)

(25:75%)

678.74 0,036*

Note: *significant

It can be seen from Table 6 that the lower percentage

of butterfly pea flower concentration had the higher

antioxidant activity than the higher percentage of

butterfly pea flower concentration. However, the

lower percentage of lemon fruits concentration had

lower antioxidant activity or vice versa. From Table

7, the antioxidant activity of the F3 formula was

7,594.55±172.80 ppm, significantly higher than the

F2 formula (8,267.80±130.63 ppm, p=<0.001) and F1

(11,287.49±123.70 ppm, p=<0.001).

4 DISCUSSION

Herein, we demonstrated that combination of

butterfly pea flower and lemon beverages had

different total flavonoids and anthocyanin levels and

antioxidant activity from butterfly pea flower or

lemon fruits alone. The total flavonoids levels and

antioxidant activity in the F3 formula were higher

than the F1 and F2 formulas. While, the anthocyanin

levels in the F1 formula were higher than the F2 and

F3 formulas.

Our findings indicated that the F1 formula

contained 75% butterfly pea flower and 25% lemon

fruits to have low total flavonoids levels and

antioxidant activity or vice versa in the F3 formula.

The posible cause of low total flavonoids levels and

antioxidant activity is come from the solvent that we

used mineral water to dissolve dried butterfly pea

flowers. In general, flavonoids compounds are

difficult to dissolve in water and have to use organic

solvents like ethanol or methanol. According to

Styawan et al., (2020) stated that there was 4.65×10

ppm total flavonoid contained in methanol extract of

butterfly pea flower. Rahayu et al., (2021) also stated

that there was 5.94×10

ppm total flavonoids level

contained in etanol extract of butterfly pea flower

(Rahayu et al., 2021). Same as Andriani and

Murtisiwi (2020), antioxidant activity IC50 in ethanol

extract of butterfly pea flower was very strong,

namely 41.36 ±1.191 ppm (Andriani & Murtisiwi,

2020).

Flavonoids were the major phytochemicals in

lemon juice. Hesperidin and hesperetin levels in

lemon juice were 85.77±0.03 ppm and 26.94±0.30

ppm, respectively (Liu et al., 2016). The lemon juice

also contains the flavanone group, including:

hesperidin, neohesperidin, naringin and eriocitrin

(Klimek-szczykutowicz et al., 2020). Therefore, the

total flavonoids levels and antioxidant activity in the

F1-F2 beverages come from the present of flavonoids

and flavanone compounds in lemon juice. It suggests

that a sinergistic effect of both butterfly pea flower

and lemon fruits does not occur in our research study.

In contrast to the total flavonoids and antioxidant

activity, the F1 formula has higher anthocyanin levels

(424.41±25.75 ppm). The lower in anthocyanin

occurred along with the lower in the composition of

dried butterfly pea flower brew in the beverage. It was

because the anthocyanin contents were known to be

only found in butterfly pea flowers and not in lemons.

According to Andriani and Murtisiwi (2021), the

extract of butterfly pea flowers had a very strong

antioxidant activity is probably due to contained the

phenolic contents. The antioxidant mechanism of

ICSDH 2022 - The International Conference on Social Determinants of Health

phenolic compounds is based on an oxidation-

reduction reaction and act as reducing agents

(Andriani & Murtisiwi, 2020). According to Utari et

al., 2017, the phenolic compounds contained in the

ethanol extract of rosela flower also had strong

antioxidant activity (Utari et al., 2017). Phenolic

compounds are semi-polar so they are not easily

soluble in water (Rondonuwu et al., 2017).

Anthocyanins are natural pigments contains

glycosides (3-O-β-glucosidase) that known have

strong antioxidant activity. Its antioxidant ability is

2x greater than other antioxidants such as catechins

and vitamin E (Sunarti, 2021). Butterfly pea flower is

known to contain delfinidine, alkaloids, saponins, and

phenols (Khaerani, 2013; Nuraini, 2014). Butterfly

pea flower extract alone were known as strong

antioxidant activity, namely IC50 95.30-132.50 ppm

(Kumar, 2019). In contrast to this experimental

results that the best antioxidant activity of the

combination butterfly pea flower and lemon

beverages were still classified as very weak. In

contrast to the results that the higher anthocyanins in

the beverage, more weak antioxidant activity

(11,287.49±123.70 ppm). This could be due to the

fact butterfly pea flower although high in

anthocyanins, had weak antioxidant activity because

that other phytochemical compounds that can

increase antioxidant activity in butterfly pea flower

do not dissolve optimally in the brewing process.

Water is a good solvent for dissolving

anthocyanin because it has a relatively similar level

of polarity (Lestario, 2017). This experiment used

drinking water as a medium for brewing dried

butterfly pea flowers at 1g/100 ml (1:100) to dissolve

881.00±24.04 ppm flavonoid compounds and

anthocyanin levels 592.50±0.45 ppm. The use of

water as a solvent in extraction was safer and

applicable for use as a formulation of food or

beverage products, although the amount of

phytochemical compounds extracted were lower

when compared to using other solvents.

Anthocyanin compounds are also easily degraded

by heating factors (Priska et al., 2018; Surianti et al.,

2019). Differences in extraction methods can also

affect the total anthocyanin levels produced.

According to Purwaniati et al., (2020) which stated

that dried butterfly pea flowers brewed at a

temperature of 50-80 degrees Celsius without

knowing the length of boiling time produced

anthocyanin levels 2-3 times higher, ranging from

1,365±0.77 ppm to 1,487±0.62 ppm but total

flavonoids were not examined (Purwaniati et al.,

2020).

Dried butterfly pea flower brew was richer in

anthocyanin levels than fresh one. It was proven by

another experiment stated 10 petals of fresh butterfly

pea flower that was brewed by water in 250 ml with

a temperature 100

Celsius for 30 minutes had

anthocyanin levels were about 510 ppm (Handito et

al., 2022). Same as Pramitasari et. Al (2022), 2 g fresh

butterfly pea flower was brewed with a temperature

Celsius had lower anthocyanin levels, namely

486±0.95 ppm (Pramitasari et al., 2022). They were

lower than this experimental result although it had

been assisted with tartaric acid in the extraction

process. There was 592,50±0,45 ppm from dried

butterfly pea flowers which was brewed in 100 ml

with lower temperature and shorter time.

Although the extraction of anthocyanin was better

by boiling at a higher temperature, but the butterfly

pea flower which also contains high flavonoids can

potentially be damaged during the process so that the

extraction method by brewing was the best alternative

to maintain the flavonoid content of butterfly pea

flower. Anthocyanin in butterfly pea flower are

known to be more extractable at temperatures ranging

from 50-80 degrees Celsius (Purwaniati et al., 2020).

Boiling dried butterfly pea flower with lower

temperature may be needed to extract more

phytochemical compounds.

However not synergistic, combination of butterfly

pea flower and lemon contained more total flavonoids

than butterfly pea flower or lemon alone (Table 2).

The type of flavonoids between butterfly pea flower

and lemon were different so that it enriched the

beverages. Each type of flavonoids have different

functions so that they will be expected to optimize

health and aim for many diseases. Futher research

about other phytochemical compounds in the

beverage is needed.

The limitation of our study is we did not

investigate the phytochemical contents in all formula

beverages, which influent the antioxidant activity.

Formulas F3 (25:75%) has better potency than other

formulas as functional beverages because they

contain more odd flavonoids and antioxidant

activities, but lowest in anthocyanin levels compared

to other formulas. The formulas have the potential to

be investigated further regarding another

phytochemical compounds and the effect of its

administration on health.

5 CONCLUSION

Combination of butterfly pea flower and lemon

beverage in the F3 formula (25:75%) has higher total

Enhancement of Flavonoid and Anthocyanin Levels of Herbal Beverage Containing Butterﬂy Pea Flower and Lemon Fruit to Improve

Anti-oxidant Activity

flavonoids levels (1.391.5±72,83 ppm) and

antioxidant activity (7.594,55±172,80 ppm) than in

the F1 and F2 formulas. Meanwhile, the anthocyanin

levels in the F3 formula is lower than F1 and F2

formulas. Futher research is needed to examine

phytochemical compounds for this beverage and

other treatment of dried butterfly pea flowers like

boiling with mineral water.

STATEMENT

There are no conflicts of interest in this research.

ACKNOWLEDGEMENTS

The researcher would like to thank the laboratory

assistants of the Food and Nutrition Laboratory of the

Faculty of Agriculture, Universitas Sebelas Maret and

the Food and Nutrition Studies Laboratory (PSPG) of

Gadjah Mada University who have assisted

researchers in the phytochemical examination

process.

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