Activity of Rose Flower Extract and Resepthakulum as Antioxidant

and Anti-tyrosinase

Lobianna Nadeak, Ermi Girsang, Chrismis Novalinda Ginting, and Linda Chiuman

University of Prima Indonesia

Keywords: Rose, Antioxidant, Anti-Aging, Anti-tirosinase.

Abstract: Rose (rosa canina) is one of the flowers that are much in demand by the community because besides being an

ornamental flower it can also be used as a cosmetic base material. Rose also contains a variety of substances

that are treated as antioxidants, and free radical scavenging. The antioxidant activity test in this study used

the parameters of diphenyl picrylhydrazin (DPPH) trapping activity and tyrosinase enzyme inhibition. Rose

petals and base of rose contain flavonoids, phenols, tannins, triterpenoids, and alkaloids. At the same

concentration of sample between the petals and the base of the rose produces different DPPH trapping

activities, where the DPPH trapping activity on the rose petals was stronger than the base of the rose. IC

value of rose petals <50 μg / ml while the base of rose > 50 μg / ml. It was seen that the effect of increasing

tyrosinase inhibition was due to an increase in the concentration of the group extract and base of rose flowers,

and the results were that at a concentration of 100 µg / ml there was a peak anti-tyrosinase activity.

1 INTRODUCTION

The characteristics of decreased beauty due to aging

of the skin in humans are rough, dull skin surface, the

appearance of brown spots, the appearance of skin

wrinkles, weak skin flexibility which must occur in

all human beings (Widowati, et.al., 2016). Excessive

exposure to ultraviolet (UV) light increases the

contribution of free radicals known as Reactive

Oxygen Specices (ROS). This certainly affects the

process of melanogenesis in the skin catalyzed by the

enzyme tyrosinase. This enzyme regulates skin

pigmentation through melamine synthesis. Increased

UV radiation will increase melanin synthesis, which

causes the risk of pigmentation or melanoma

disorders (Lai, Wichers, Soler-Lopez & Dijkstra,

2018).

Bioactive compounds that exist in plants such as

isoflavones, anthocyanins, and katesin have strong

antioxidant activity against free radicals called

neutralizing excessive Reactive Oxygen Species

(ROS). The body has enzymatic and non-enzymatic

antioxidant defense systems. The antioxidant

enzymes are superoxide dismutase, catalase, and

glutathione peroxidase. While non-enzymatic

antioxidants are glutathione, tocopherol (Vitamin E),

Vitamin C, b-carotein and selenium (Shalaby and

Shanab, 2013; Ismail et al, 2020). Lately antioxidants

have become something of interest in the medical

world, known to have an effect on preventing

premature aging (anti-aging) against free radicals

(Garg, Khurana & Garg, 2017).

Rose plant (Rosa canina) contains vitamins (B, P,

PP, E, K, and C), flavonoids, carotene, carbohydrates,

and organic acids which most of these substances

have properties as anti-oxidant, anti-inflammatory,

free radical scavenging inhibits the oxidation process

(Masek, Latos, Chrzescijanska & Zaborski, 2017).

Flavonoid acid as an antioxidant and anti-tyrosinase

is very beneficial against skin pigmentation (Zuo,

et.al., 2018). Tyrosinase or polyphenol oxidase is an

oxidireductase that plays a role in melanin

biosynthesis and is the main pigment in hair, eyes and

skin. The reaction of the tyrosinase enzyme with the

L-DOPA substrate can produce an orange color.

Inhibition of the activity of the tyrosinase enzyme is

characterized by a reduction in the orange color that

is formed or the result of a color reaction becoming

more clear while simultaneously marking the

occurrence of antioxidant activity (Fais, et al., 2009).

The tyrosinase enzyme is an enzyme responsible

for skin darkening or melanogenesis. Tyrosinase in

humans is a complex protein and is precisely folded,

expressed and undergone post-translational

modifications including Heavy glycolysation

Ginting, C., Nadeak, L. and Chiuman, L.

Activity of Rose Flower Extract and Resepthakulum as Antioxidant and Anti-tyrosinase.

DOI: 10.5220/0010285800170022

In Proceedings of the International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical (HIMBEP 2020), pages 17-22

ISBN: 978-989-758-500-5

(Zolghadri, et al., 2019). Diphenyl picrylhydrazin

(DPPH) compound is a free radical that is stable in

aqueous or methanol solution and has a purple color

(indicated by the absorption band in the methanol

solvent at a wavelength of 515-520 nm). The DPPH

has properties that are sensitive to light, oxygen, and

pH, but are stable in the form of radicals so that it is

possible to measure an accurate antioxidant activity.

The antioxidant compounds will release hydrogen

atoms to form radical antioxidant compounds. The

DPPH which is a free radical that reacted with

antioxidant compounds to form non-radical DPPH

(Widowati, et.al., 2016). Mini rose showed the

greater antioxidant activity in the ferric reducing

antioxidant power (FRAP) and DPPH tests before

digestion in vitro and together with cosmos as a

source of phenolics with good antioxidant activity (de

Morais, et.al., 2020). Dry rose tea can be used as a

functional food to be a source of natural antioxidants

(Kart & Çağındı, 2017).

Previous studies have shown that phytochemical

compounds contained in Rosa Damascena flower

petals consist of alkaloids, flavonoids, tannins,

carbohydrates, and amino acids (Tatke, Satyapal,

Mahajan & Naharwar, 2015). It has also been found

that rose petal extract contains high anthocyanins,

flavonoids, polyphenols (Lee, et.al., 2018). Unlike

the results of the phytochemical tests in this study,

where the rose petals also contain triterpenoids and

terpenoids.

Measuring the effectiveness of a compound in

biological or biochemical functions capable of

inhibiting the oxidation process by 50% (IC

) was

classified in several groups including <50 µg µg per

milliliter (very strong); 50-100 µg µg per milliliter

(strong); 101-150 µg µg per milliliter (moderate); >

150 µg µg per milliliter (weak) (Budaraga, Marlida &

Bulanin, 2016). The IC

value of extracted by DPPH

method on black soybean and daidzein was 116.52 µg

/ mL and 109.34 µg / mL, repectively, which means

it has moderate antioxidant strength (Kuswanto,

2018). Ethanol extract of cocoa beans and kojat acid

can be used as inhibitors of enzymaticrosinase

(Kurniasari, Djajadisastra & Anwar, 2018). Oil

obtained from Nigela Sativa seeds (known as black

cumin) is also often used as an anti-oxidant and anti-

inflammatory (Bordoni, et.al., 2019). Manga waste

(skin, seed coat, seeds) from the Colombian manga

cultivar is a source of phenolic compounds that can

be used as antioxidants and free radical cleaners

(Castro-Vargas et al., 2019).

The antioxidant activity test of Rosa damascena

rose petal extract was using standard ascorbic acid

(iron reducing power test) and showed the highest

antioxidant in the cream formula (Safia, et.al., 2019).

The results show that rose extract has good potential

for cosmetic development. Rose oil has the strongest

antioxidant effect and a mixture of Rose oil, bergamot

and patchouli (RBP) with a volume ratio of 7: 2: 1

produces the strongest antioxidant effect on the

DPPH and ATBS Test [2,2-azinobis (3-

ethylbenzothiazoline-6 sulphonic acid)]

(Wongsukkasem, e.al., 2018). In that study also found

that rose oil and bergamot have an antityrosinase

activity around 28 ± 14.2% and 21 ± 10.7%.

However, the study did not specifically explain

whether there were differences in antioxidant

concentrations between the base and rose petals.

2 METHOD

This research is an experimental laboratory study

with data collection (random sampling) using

samples of rose extract and receptacle. The study was

conducted from July-September 2019 at the

Biomolecular Laboratory and Biomedical Research

Center (Aretha Medika Utama). The sample used was

a rose obtained from the Source Seed Management

Unit, Research Institute for Miscellaneous Plant

Flowers in Malang and has gone through a process of

determination. Rose was washed and dried in the sun

to dry milled and extracted by maceration technique

using 70% ethanol solvent for 3 days at room

temperature. Next, the marinade was filtered to

separate the filtrate and the residue. The obtained

filtrate was evaporated with

amotor unit that rotates

the evaporation flask in 50

celcius degrees, so that a

solid extract was received.

Rose petals has 1400 g net weight, simplicia

powder of rose petals was 250 grams and the basic

wet weight of rose flower was 700 g processed into

simplicia powder, base of rose flowers about 90

grams each dissolved in 70% ethanol for 3 days by

maceration method, so the extract of rose petal about

88.56 and 2.72 grams of rose base extract.

The tools and materials used in the

phytochemical, DPPH trapping, and tyrosinase tests,

respectively, are given in Table 1. Phytochemical

tests are used to identify phenol compounds, steroids

/ triterpenoids, saponins, tannins, terpenoids,

flavonoids, and alkaloids found in the lids, and bases

roses.

Identification of phenol was carried out by

dissolving extracts of petals and rose base 10 mg in

ddH2O about 5 ml then adding 500% FeCl

solution

about 500 µl. It is known that the sample solution

contains phenol group compounds if one of the

HIMBEP 2020 - International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical

primary colors is red or blue or the secondary colors

are green, purple, or black.

The process of identifying steroids / triterpenoids

was done by dissolving 10 mg into a drip plate,

adding glacial acetic acid until submerged for 10-15

minutes, then adding one drop of concentrated

. If the solution produces blue green, this

portion of the population contents the more chemical

elements of the steroid class. Whereas if the solusion

produces purple / red / orange color, this portion of

the population contents the more chemical elements

of the triterpenoid group.

Table 1: Materials and tools.

Materials Tools

1. Phytochemical Test

Rose Petal Extract; Rose

base extract; FeCl3 (1% in

ddH2O) (Merck,103943);

Aquades (ddH2O); HCl 2 N

(Merck 1090631000); HCl

1 N (Merck 109057);

H2SO4 pekat (Merck

1007310510); Amil alcohol

(Merck 1009751000);

Vanilin (Sigma-Aldrich,

W310727); Mg/Zn powder

(Merck 1058151000,

1087560250); Dragendorff

reagents (potassium iodide

Merck 207969; and bismuth

nitrate Merck 248592

)

Test tube; Spatula; Mikropipet (1-10 µl,

50- 200 µl, 100-1000 µl) (Eppendorf);

Tips 10, 200, 1000µl (NEPTUNE);

Waterbath (Hanyang); and Drip plate..

2. DPPH Trapping Test

Rose Petal Extract; Rose

base extract; 2,2 Diphenyl-

1-picrylhydrazyl (DPPH)

(Sigma D9132); Methanol

absolute (Merck

1060092500); DMSO

(Merck 1029522500); and

Akuades (ddH2O).

pH meter (OHAUS Starter300 portable);

Erlenmeyer Tube; Spatula; Magnetic

stirrer and hot plate (Thermo Fisher

Scientific); Multiskan Go Reader

(Thermo Fisher Scientific, 1510);

Incubator (ESCO, IFA-32-8);

Micropipette (1-10 µl, 50- 200 µl, 100-

1000 µl) (Eppendorf); 96 well-plate

(Costar, 3596); Falcon tube 15 ml (SPL,

50015); Falcon tube 50 ml (SPL,

50050); Analytical Balance (AXIS);

Tube Effendorf 1,5 ml (SPL, 60015-1);

Vortex (WiseMix, VM-10); and Tips (1-

10 µl, 50- 200 µl, 100-1000 µl)

(

Borusil

)

3. Tyrosinase Test

Rose Petal Extract; Rose

base extract; Potasium

dihydrogen phosphate

(Merck 104873);

Dipotasium hydrogen

phosphate(Merck 105104);

Tyrosinase from Mushroom

(Sigma T3824); L-DOPA

(3,4-Dihydroxy-L-

phenylalanine) (Sigma

D9628); Potasium Hydroxyl

(Sigma P5958); and

Aquades (ddH2O).

pH meter (OHAUS Starter300 portable);

Erlenmeyer Tube; Spatula; Magnetic

stirrer and hot plate (Thermo Fisher

Scientific); Multiskan Go Reader

(Thermo Fisher Scientific, 1510);

Incubator (ESCO, IFA-32-8);

Micropipette (1-10 µl, 50- 200 µl, 100-

1000 µl) (Eppendorf); 96 well-plate

(Costar, 3596); Falcon tube 15 ml (SPL,

50015); Falcon tube 50 ml (SPL, 50050);

Analytical Balance (AXIS); Tube

Effendorf 1,5 ml (SPL, 60015-1); Vortex

(WiseMix, VM-10); and Tips (1-10 µl,

50- 200

l, 100-1000

)

(

Borusil

)

Identification of saponins in the sample is the

presence of foam which is always stable, after going

through the process of dissolving 10 mg of petals and

rose flower base extract using ddH2O in a heat-

resistant glass container, heated to boiling for 5

minutes, filtered, stirred vigorously and the addition

of 1 N hydrogen chloride solution. The identification

of tannins can be done by entering a 10 mg sample

into a heat-resistant glass container containing 2 N

hydrochloric acid with a volume of 2ml. Heating is

carried out in a water medium which lasts for half an

hour. After that, add the type of alcohol pentanol

OH) with a volume of 500 µl. The pentanol

layer will produce a tannin group compound if one of

the layers is orange or red.

Terpenoids are identified by entering a 10 mg

sample into a drip plate, adding vanillin to taste,

adding concentrated H

to one drop and then

homogenizing, if it produces a purple color then the

sample contains compounds of terpenoid class.

Identification of flavonoids was carried out by

dissolving a sample of 10 mg of petal extract and rose

flower base in a test tube containing 2 N hydrogen

chloride. Add sufficient magnesium or zinc, then heat

for 5-10 minutes, cool and filter. After that, add

pentanol with a volume of 1 ml. Extract samples and

rose petals will produce flavonoid class compounds if

the sample gives either red or orange color.

Identification of alkaloids by dissolving samples

of rose petals and base extracts in 5 ml ddH2O

evaporated in a water bath. After producing a

precipitate, immediately add 5 milliliters of 2N

hydrochloric acid. The resulting solution is divided

into 2 test tubes. The first tube is called a comparator

by inserting 3 drops of 2N HCl. While the second tube

solution is transferred as much as one drop to the drop

plate, then give 3 drops of Dragendorff reagent.

Identify alkaloids (+) if they form an orange

precipitate.

A total of 200 µL DPPH 0.077 mmol in methanol

was added with a rose sample extract and 50 µL

receptacle each on the microplate. The mixture was

incubated at room temperature for 30 minutes to

obtain the absorbance value at a wavelength of 517

nm using a microplate reader. For negative control,

250 mL DPPH was used, while for blanks, 250 mL

absolute DMSO was used (Widowati, et.al., 2016).

The antioxidant activity by DPPH (%) method is:

DPPH trapping activities (%) =

absorbance of the sample

absorbance control

x 100

(1)

Zulghadar described that the method of inhibiting

tyrosinase enzyme activity was carried out with minor

changes (Zolghadri, et.al., 2019). A mixture of

aqueous solution consisting of 20 µL samples of rose

extract and receptacles (concentration 100 µg / mL;

50 µg / mL; 25 µg / mL; 12.50 µg / mL; 6.25 µg / mL;

3.13 µg / mL ), 20 µL of the Tyrosinase from

Mushroom enzyme (125 U / mL), and 140 µL of

Activity of Rose Flower Extract and Resepthakulum as Antioxidant and Anti-tyrosinase

potassium phosphate buffer (20 mM, pH 6.8) were

incubated at room temperature during a quarter of an

hour. Besides that, it was also prepared for controls

containing only 20 µL enzymes and 160 µL

phosphate buffer and blanks which only contained

160 µL phosphate buffer and 20 µL samples. Further,

the solution mixture was added as much as 20 µL of

L-DOPA substrate (1.5 mM) and re-incubated at

room temperature for 10 minutes. Absorbance was

measured using a wavelength of 470 nm. The

percentage of inhibitory activity was calculated using

the following formula:

% inhibition =





 100 

(2)

where C is the enzyme absorbance activity without a

sample, and S is the enzyme absorbance activity by

the addition of the tested sample.

The obtained data the experiment was processed

using the SPSS program with the One-Way ANOVA

test and continued by the Post Hoc Test using the

Tukey HSD test with a confidence level of 95% (α =

0.05). The DPPH and anti-tyrosinase activity test

results were followed by an analysis of the linear

regression equation to determine the value of

Inhibition Concentration 50 (IC

). Overall, the

stages of the rose extraction process can be seen in

Figure 1.

Figure 1: Extraction of the Rose petals and base of roses

Procedure: (a) separation, (b) dry separation, (c) grinded,

(d) immersed in 70% ethanol, (e) accommodated ethanol,

(f) ethanol filtrate evaporation, (g) 70% ethanol extraction

yield.

3 RESULTS AND DISCUSSION

It was found that phytochemical screening for rose

petals and base of rose contained flavonoids, phenols,

tannins, triterpenoids and alkaloids, without saponins.

Rose petals contain terpenoids but not for base of the

rose. Antioxidant activity by trapping DPPH in rose

petal extract was always higher than rose base extract

at various concentrations. This result can be seen in

Table 2, where the data is presented in the form of an

average ± standard deviation. The difference in the

percentage of DPPH and anti-tyrosinase trapping

activity at each concentration (expressed in µg / ml)

indicates that F count> F table with p <0.001 <α =

0.05. The total antioxidant activity of rose petals and

base of rose extracts will increase with the increase of

theconcentration. At the maximum concentration,

rose extract showed the highest antioxidant activity.

Table 2: Statistics of Antioxidant Activity of Rose Petals

Extract and Base of Rose (Average, Post Hoc Test Results

of Tukey HSD Test).

Final

Concentration

(µg/ml)

DPPH arrest rate (%)

rose petals base of rose

6.25 44.36±0.92

33.91±0.37

12.50 48.17±0.80

38.00±2.44

25.00 55.03±0.50

44.14±0.49

50.00 62.59±1.50

50.32±0.42

100.00 76.76±0.32

64.80±0.58

200.00 103.98±0.03

90.66±0.74

Anti-tyrosinase activity in base of rose (br) was

higher than rose petal (rp) extracts, except at a

concentration of 6.25 µg / ml. This can be seen in

Table 3. The highest anti-tyrosinase activity of roses

at a concentration of 100 μg / mL.

Table 3: Statistics of Inhibitory Activity of Tyrosinase

Extract of Rose Petals (rp) and Base of Rose(br) (Mean,

Post Hoc Test Results of Tukey).

Final

Consentration

(µg

/ml

)

Avera

e t

rosinase inhibition

(

)

rp br

3.125 20.56±1.59

20.95±1.69

6.25 24.59±0.72

23.95±1.16

12.5 28.43±2.04

29.00±2.35

25 35.03±1.61

36.40±1.84

50 44.94±1.40

45.65±0.75

100 66.33±0.72

69.25±1.51

Antioxidant activity by trapping DPPH in rose

petal extract is higher than base of rose extract. This

can be seen in Table 4 where the average IC

value

of rose petal extract was 14.89 µg / mL; while in the

basic extracts of roses, the average IC

value was

52.81 µg / mL. In the anti-tyrosinase (A-TS) activity

test on base of rose, it is more effective than rose

petals, it can be seen that IC

values are 62.27 μg /

mL and 58.66 μg / mL for rose petals and base of rose,

reapectively.

HIMBEP 2020 - International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical

Table 4: IC

Value of DPPH Trapping and Anti-tyrosinase

Activity from rose petal and base of rose extracts.

Sample

Regression

equation

(µg/ml)

Average

(µg/ml)

A-

rp 1 y =

0.2

45.

y =

0.4

427

x +

22.

529

0,9

933

0.9

941

14,

62,

14.

rp 2 y =

0.2

45.

y =

0.4

576

x +

21.

795

0,9

859

0.9

759

14,

61,

rp 3 y=

0.3

45.

y =

0.4

596

x +

20.

998

0,9

904

0.9

904

15,

63,

br 1 y =

0.2

x +

35.

y =

0.4

765

x +

22.

628

0,9

889

0.9

848

50,

57,

52.

br 2 y =

0.2

34.

y =

0.4

899

x +

21.

0,9

921

0.9

804

53,

57,

br 3 y =

0.2

34.

y =

0.4

772

x +

20.

863

0,9

920

0.9

946

45,

61,

4 CONCLUSION

Comparison of antioxidant activity through DPPH

trapping based on IC

value of rose petal extract has

a value of 14.89 μg / ml and rose base extract has

52.81 μg / ml. This shows that the rose petals are

stronger than the base of the rose. The IC

value of

the antitirosinase test results on rose petal extract and

rose base have antitirosinase activity of 62.27 μg / ml

and 58.66 μg / ml, respectively. These results indicate

that the base of the rose is more effective than rose

petals

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https://doi.org/10.1186/s13020-018-0206-9.

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HIMBEP 2020 - International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical