Potential of Black Tea (Camellia Sinensis (L.) O. Kuntze) Extract as

Anti-oxidant and Skin Anti-aging

Wahyu Widowati

1,* a

, Rita Tjokropranoto

, Cindy Damayanti

, Hanna Sari Widya Kusuma

Tri Handayani

and Rizal Rizal

2,3 f

Faculty of Medicine, Maranatha Christian University, Jl. Surya Sumantri No. 65, Bandung, West Java, Indonesia

Biomolecular and Biomedical Research Center, Aretha Medika Utama, Jl Babakan Jeruk II No. 9, Bandung,

West Java, Indonesia

Biomedical Engineering, Department of Electrical Engineering, Faculty of Engineering, Universitas Indonesia,

Depok, West Java, Indonesia

mbaktrihandayani@gmail.com, rizal_biotek@yahoo.com

Keywords: Black Tea, Anti-oxidant, Anti-aging, Free Radicals, Collagen.

Abstract: Background: Skin aging is characterized by features such as wrinkling, loss of elasticity, laxity, rough

textured appearance, and phenotypic changes in cutaneous cells. Skin aging treatment usually with a

synthetic compound with unknown side effects but with herbal such as black tea, these side effects will be

minimalized. Objective: This research was conducted to evaluate the qualitative phytochemical screening

assay, total phenolic and flavonoid contents, anti-oxidants, and skin anti-aging properties of black tea

extract (BTE). Method: This qualitative phytochemical content using the Farnsworth modified method.

Total phenol content was calculated using gallic acid equivalent (GAE), and total flavonoid content was

calculated using quercetin equivalent (QE). The anti-oxidant properties using 2,2 diphenyl 1 picrylhydrazyl

(DPPH), 2,2′-Azinobis (3- Ethylbenzthiazoline-6-Sulfonate) (ABTS), hydrogen peroxide (H2O2)

scavenging activities. The anti-aging properties were assayed using elastase and collagenase inhibition

activities. Results: BTE contained terpenoids, triterpenoids, phenols, flavonoids, tannins. BTE contained

phenol 52.81 μg GAE/mg, flavonoids 10.96 QE/mg. The IC50 value of DPPH, ABTS, H2O2 scavenging

activities was 15.29; 88.18; 17.21 μg/ml respectively. The IC50 value of elastase and collagenase inhibition

was 31.34; 123.74 μg/ml respectively. Conclusion: BTE has highly active and active anti-oxidant and is also

less active and moderately active in skin anti-aging activities.

https://orcid.org/0000-0002-5401-7794

https://orcid.org/0000-0002-3077-9057

https://orcid.org/0000-0003-4181-5559

https://orcid.org/0000-0002-7422-0036

https://orcid.org/0000-0001-9186-9841

https://orcid.org/0000-0003-2783-0672

Corresponding author

1 INTRODUCTION

Aging is a normal physiological process experienced

by all creatures. Skin is the organ that is exposed to

the outer environment so skin suffers from both

intrinsic and extrinsic aging factors. Skin aging can

be characterized by wrinkling, loss of elasticity,

laxity, and rough-textured appearance (Zhang &

Duan, 2018). There are two kinds of factors

inducing skin aging intrinsic and extrinsic factors.

Intrinsic factors are mainly due to genetic and

metabolic factors (Mancini et al., 2014). Collagenase

is the enzyme to cleaving native collagen under the

physiological condition in vivo and in vitro

(Holmbeck & Birkedal Hansen, 2013; Widowati et

al., 2016; 2017; 2018a, 2018b). While elastin plays a

role in maintaining the elasticity of the skin that can

be degraded by enzymes elastase. While the factors

extrinsic including, stress, lifestyles such as smoking

and drinking alcohol, exposure to sun or UV rays.

Premature aging is most often caused by air

pollution and photoaging or UV light. The effects of

Widowati, W., Tjokropranoto, R., Damayanti, C., Kusuma, H., Handayani, T. and Rizal, R.

Potential of Black Tea (Camellia Sinensis (L.) O. Kuntze) Extract as Anti-oxidant and Skin Anti-aging.

DOI: 10.5220/0010744400003113

In Proceedings of the 1st International Conference on Emerging Issues in Technology, Engineering and Science (ICE-TES 2021), pages 65-73

ISBN: 978-989-758-601-9

photoaging exposure in the long term will result in

an increased risk of premature aging caused by

Reactive Oxygen Species (ROS) which can affect

collagenase and elastase enzymes and levels of anti-

oxidants in the body. That will make the appearance

of wrinkles and dark stains on the skin arise.

(Widowati et al., 2016; 2017; 2018a, 2018b).

To ward off free radicals in the body, humans

can produce anti-oxidant enzymes, e.g., glutathione

peroxidase (GPX), catalase (CAT), and superoxide

dismutase (SOD) (Yadav et al., 2016), but the

amount is still less effective in overcoming oxidative

stress that happens to the body. Many people

consume anti-oxidants and skin anti-aging synthesis

as cosmetics or creams, but over long periods can

cause side effects such as hyperpigmentation or

malignancy of the skin. So it is sought alternatives

from natural ingredients which have anti-oxidant

and skin anti-aging activities such as pineapple

(Jusri, 2019), dragon fruit (Liana, et al., 2019),

jasmine flowers (Widowati et al., 2018a), white rice

(Widowati et al., 2016), rosella flowers (Widowati et

al., 2017), and black tea extract have activity anti-

oxidants (Widowati et al., 2015). Tea has a bioactive

component, such as polyphenols. In general, the

classification of polyphenols exists 2, namely

phenolic acids and flavonoids (Sudaryat et al.,

2015). The function of flavonoids is to protect the

body from damage caused by ROS, inhibit

degenerative diseases, and inhibit the activity of lipid

peroxidase (Sudaryat et al., 2015). Tea also contains

theophylline, tannins, vitamin B complex, C, E, K.12.

The black tea contains bioactive compounds

alkaloids, flavonoids and tannins, phenols, saponins,

and steroids that are thought to have anti-oxidant

activity, anti-collagenase, and anti-elastase (Sudaryat

et al., 2015, Widowati et al., 2015).

This research was conducted to find out the

content of various compounds phytochemicals of

black tea extract obtained commercially

manufacture. The study also measured anti-oxidant

activity including H

, DPPH, and ABTS

scavenging activity, and skin anti-aging activities

including anti-collagenase and anti-elastase of black

tea extract (BTE). This research was conducted for

qualified cosmetic preparation to use certified

extract based on good manufacture practice (GMP).

2 MATERIALS AND METHODS

2.1 Sample Preparation

BTE was obtained from Indesso (SP-766-3). The

BTE using water solvent and followed by the spray

drying process, moisture content 7.0%, turbidity ≤35

FAU, polyphenol content ≥ 15%, soluble in water,

ingredient: BTE and maltodextrin (Certificate of

Analysis by Indesso, Jakarta, Indonesia).

2.2 Phytochemical Analysis

Phytochemical analysis was used to determining the

secondary metabolite content of BTE. The

phytochemical analysis in this research consisted of

flavonoid, saponin, phenolic, tannin, alkaloid,

steroid/triterpenoid, and terpenoid content of BTE.

2.2.1 Flavonoid Content

A 10 mg extract of BTE dissolved in HCl 2 N in the

reaction tube. In the mixture, Mg/Zn was added

sufficiently, then was heated for 5-10 minutes,

cooled down filtered, and added 1 ml of amyl-

alcohol. If red/orange color formed then the sample

contained flavonoid (Widowati et al., 2018a;

Prahastuti et al., 2019; Prahastuti et al., 2020).

2.2.2 Saponin Content

A 10 mg of BTE dissolved in ddH2O in the reaction

tube. The sample heated until boiling for 5 minutes

then filtered and shake strongly before added HCl 1

N. If the bubble still formed and still exist after HCL

1 N was added, then the sample contained saponin

(Widowati et al., 2018a; Prahastuti et al., 2019;

Prahastuti et al., 2020).

2.2.3 Phenolic Content

A 10 mg of BTE dissolved in 5 ml ddH

O. Briefly

500 µl FeCl

1% added into mixture. If

green/red/purple/blue/black color formed, the

sample contained phenol (Widowati et al., 2018;

Prahastuti et al., 2019; Prahastuti et al., 2020).

2.2.4 Tannin Content

A 10 mg of BTE dissolved in 2 ml HCl 2N in the

reaction tube. The mixture was heated in the water

bath for 30 minutes, cooled down, then 500 µl of

amyl alcohol was added. If there was an orange/red

color in the amyl-alcohol layer, then the sample

contained tannin (Widowati et al., 2018; Prahastuti

et al., 2019; Prahastuti et al., 2020).

2.2.5 Alkaloid Content

A 10 mg of BTE extract dissolved in 5 ml ddH

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

and evaporated in the water bath. The residue from

the evaporation dissolved in 5 ml HCl 2N. Then the

mixture is divided into two reaction tubes. The first

tube added 3 drops of HCl 2N as a blank. The

mixture from the second tube took 1 drop into the

dropping plate, then 3 drops of Dragendorff mixture

were added. If the orange solid formed, then the

sample contained alkaloids (Widowati et al., 2018a;

Prahastuti et al., 2019; Prahastuti et al., 2020).

2.2.6 Steroid/Triterpenoid Content

Glacial acetic acid is added into 10 mg BTE in the

dropping plate, let the mixture for 10-15 minutes. In

the mixture added 1 drop of concentrated H2SO4. If

there was greenish-blue color formed then the

sample contained steroid, but if there was a

purple/red/orange color formed then the sample

contained triterpenoid (Widowati et al., 2018a;

Prahastuti et al., 2019; Prahastuti et al., 2020).

2.2.7 Terpenoid Content

Vanillin was added into 10 mg BTE sufficiently into

the dropping plate. 1 drop of concentrated H2SO4

was added then homogenized. If there was a purple

color formed, then the sample contained terpenoid

(Widowati et al., 2018a; Prahastuti et al., 2019;

Prahastuti et al., 2020).

2.3 Total Phenolic Content

A 15 μl of a sample (BTE and gallic acid) was

loaded into the sample and blank wells. Briefly, 75

μl Folin-Ciocalteu 10% reagent was added into

sample wells. 60 μl Na2CO3 7.5% added to sample

wells. Into the blank well, 150 μl DMSO 10% was

added then incubated for 10 minutes at 50oC.

Absorbance was measured with a microplate reader

at λ = 760 nm. Gallic acid (GA) is used as a standard

to determine the phenolic concentration (Widowati

et al., 2018a; Prahastuti et al., 2019; Prahastuti et al.,

2020).

Linear standard equation: y = ax + b (1)

Total Phenol =

𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑏

𝑎

(2)

2.4 Total Flavonoid Content

Briefly, 75 μl of a sample (BTE and Quercetin)

added to the sample and blank wells. Amount 75 μl

AlCl3 2% (in glacial acetic acid 5% and methanol)

was added into sample wells. Into the blank well, 75

μl DMSO was added. Absorbance was measured

with a microplate reader at λ = 415 nm. Total

flavonoid calculated with the standard linear

equation of Quercetin (Widowati et al., 2018a;

Prahastuti et al., 2019; Prahastuti et al., 2020).

Linear standard equation:

= ax + b (3)

Total Flavonoid =

𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑏

𝑎

(4)

2.5 H2o2 Scavenging Activity

Method to determine the activity of H2O2

scavenging based on the modified method by

Prahastuti et al. (2019 and (2020). Each sample well

was contained 60 μL of BTE, 12 μL of

FeH8N2O8S2 1 mM, and 3 μL of H2O2 5 mM. The

mixture that contains 12 μL of FeH8N2O8S2 and 63

μL of DMSO was used as the negative control, while

the mixture that contains 60 μL of BTE and 90 μL of

DMSO were used as the blank solution. After H2O2

was added, the mixture was incubated in the dark

and room temperature for 5 min. The 75 μL 1,10

phenanthrolines were added into the sample and

control well and incubated again for 10 min in the

dark and room temperature. Sample absorbance was

measured at 510 nm (Prahastuti et al., 2019;

Prahastuti et al., 2020; Girsang et al., 2020).

Percentage of H2O2 scavenging activity

calculated with the equation:

% H

Scavenging Activity =

𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

x 100

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

(5)

2.6 DPPH Scavenging Activity

Briefly, 200 µl DPPH 0.077 mmol in methanol was

added into 50 µl BTE in a microplate. Mixture

incubated at room temperature for 30 minutes then

absorbance measured at 517 nm with a microplate

reader. Amount 250 µl DPPH used as negative

control and 250 µl absolute DMSO used as blank

(Prahastuti et al., 2019; Girsang et al., 2020;

Prahastuti et al., 2020; Mawarni et al., 2020). Anti-

oxidant activity of the DPPH method (%):

% DPPH Scavenging Activity =

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

x100

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏 𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

(6)

2.7 ABTS Scavenging Activity

Briefly, 2 µl sample added into 96-well plate and

then 198 µl ABTS working reagent added into the

Potential of Black Tea (Camellia Sinensis (L.) O. Kuntze) Extract as Anti-oxidant and Skin Anti-aging

sample well. 200 µl DMSO used as blank and 200 µl

ABTS working reagent used as control. Plate

incubated for 6 minutes at 37oC. The absorbance of

the sample was measured at 745 nm (Prahastuti et

al., 2019; Prahastuti et al., 2020; Girsang et al.,

2020; Mawarni et al., 2020). Anti-oxidant activity of

the ABTS method (%):

% ABTS Reducing Activity =

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏−𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏

x 100

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏

(7)

2.8 Collagenase Inhibition Activity

Method to measure the inhibition activity of

collagenase based on the method by Sigma Aldrich

with a little modification (Widowati et al., 2018a;

Utami et al., 2018 Girsang et al., 2020). Sample

mixture consist of 30 µl sample (7.81-250 µg/ml),

10 µl Collagenase from Clostridium histolyticum

(0.1 mg/ml, Sigma C8051), and 60 µl tricine buffer

(50 mM Tricine, 10 mM calcium chloride, 400 mM

sodium chloride, pH 7.5) incubated at 37oC for 20

minutes. 10 µl enzyme and 90 µl phosphate buffer

used as control and 10 µl enzyme, 80 µl phosphate

buffer, and 30 µlsample used as blank. 20 µl

FALGPA (1 mM, Sigma F5135) was added to each

well except blank. Sample absorbance was measured

at 335 nm. Inhibition activity calculated with the

equation:

% Collagenase Inhibition =

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

x100

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

(8)

2.9 Elastase Inhibition Activity

Method to measure the inhibition activity of elastase

based on the method by Sigma Aldrich and

Widowati et al. (2018a) with a little modification

(Utami et al., 2018; Girsang et al., 2020; Mawarni et

al., 2020). Sample mixture consist of 10 µl sample

(2.08-66.67 µg/ml), 5 µl elastase from porcine

pancreas (0.01 mg/ml, Sigma 45124) and 125 µl Tris

buffer (100 mM, pH 8, Pharmacia Biotech 17-1321-

01) incubated at 25oC for 15 minutes. Briefly 5 µl

enzyme and 135 µl Tris buffer used as control and

130 µl Tris buffer and 10 µl sample used as blank.

Amount 10 µl SucAla3-pNA was added to each well

and incubated at 25oC for 15 minutes. Sample

absorbance was measured at 410 nm. Inhibition

activity calculated with the equation:

% Elastase Inhibition =

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

x100

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

(9)

2.10 Statistical Analysis

All data from H2O2, DPPH, and ABTS scavenging,

collagenase, and elastase inhibition were analyzed

statistically with ANOVA and Tukey HSD Post Hoc

Test (P<0.05). Inhibitory Concentration (IC50) using

linear regression analysis. The column graph was

formed with Graph Pad 7 Prism.

3 RESULT AND DISCUSSION

3.1 Phytochemical Analysis

Phytochemical analysis was done to know what kind

and how many secondary metabolite properties were

from the sample. Even the data obtained as

qualitative data, but data can become the base to

pick the method to measure the total content of the

secondary metabolite of the sample. BTE contains

low content of alkaloid (+), not detected saponin (-),

very high content of phenol (++++), low tannin (+),

not detected steroid (-), high triterpenoid (+++), high

terpenoid (+++), and moderate alkaloid (++).

This result is slightly different from the research

done by Widowati et al. (2015) that showed black

tea extract contains medium content on flavonoid,

terpenoid, and phenol, low content on terpenoid,

tannin, and saponin, and negative content on

alkaloid and steroid. During fermentation, catechin

is oxidized and polymerized into theaflavins (TF)

and thearubigins (TR) or degraded to another form

(Das and Datta, 2019). The difference of secondary

metabolites of BTE is caused by different

environmental and management factors (Ahmed et

al., 2019) including plant genotype (Cherotich et al.,

2013; Chen et al., 2018; Mu et al., 2018), shade

(Sano et al., 2018), elevation (Han et al., 2017;

Kfoury et al., 2018), drought (Scott et al., 2019),

precipitation (Ahmed et al., 2014; Kowalsick et al.,

2014), temperature (Ahmed et al., 2019), model of

agricultural production (Ahmed et al., 2013; Han et

al., 2018), microbes (Ahmed et al., 2019), and

numerous pest insects (Scott and Orians, 2018).

Because the source of used BTE in this research was

different from previous research resulted different

secondary metabolites (Ahmed et al., 2019). The

different extraction solvents resulted in different

compound content (Yusnawan, 2013). The different

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

extraction solvents resulted from different

bioactivity (Ngo et al., 2017).

3.2 Total Phenolic and Flavonoid

Content

Total phenol activity was used to know how many

phenol contained in the sample by measure the

sample + reagent with spectrophotometry. The

phenol content of BTE is 52.81±1.38 µg QE/mg

sample. Total flavonoid activity was used to know

how many flavonoids were contained in the sample

by measure the sample + reagent with

spectrophotometry. The flavonoid content of BTE is

10.96±12.46 µg QE/mg sample.

Phenol total activity of BTE is 52.81 µg

GAE/mg extract indicated that flavonoid is the most

secondary metabolites in black tea. But the

flavonoid total activity is 11.73 µg QE/mg extract,

this result was in line with previous research that

BTE has phenol standard catechin (14.33 μg

Catechin/mg), kaempferol (4.33 μg kaempferol/mg),

myricetin (4.17 μg myricetin/mg) dan quercetin

(4.30 μg quercetin/mg) (Widowati et al., 2015).

3.3 H2O2 Scavenging Activity

BTE has anti-oxidant activity in scavenging H2O2

with a higher concentration of BTE increased the

H2O2 scavenging activity (Figure 1).

Figure 1: Anti-oxidant activity of BTE at various

concentrations against H2O2 scavenging activity.

*The data was presented as mean ± SD. The assay was

done in triplicate for each concentration. The different

letter (a.b.c.d.e) showed significant difference among BTE

concentration (P<0.05).

The IC50 value of H2O2 scavenging activity was

88.18 µg/ml, according to Marjoni and Zulfisa

(2017), BTE was categorized as active when the

IC50 value <100 µg/ml. The research was done by

Fernando and Soysa (2015) the BTE has Effective

Concentration 50 (EC50) at 91.96 µg/ml. The EC50

means the half maximal effective concentration

extract to scavenge the H2O2 that same with IC50.

The IC50 of BTE is nearly the same that means the

result is valid.

3.4 DPPH Scavenging Activity

BTE has anti-oxidant activity in scavenging DPPH

with higher concentration BTE increased the DPPH

scavenging activity (Figure 2). The IC50 value of

DPPH scavenging activity of BTE is 15.29 µg/ml

which means with 15.29 µg/ml extract can inhibit

50% of DPPH become lost its radical properties, it

was categorized highly active anti-oxidant (Marjoni

and Zulfisa, 2017).

Figure 2: Anti-oxidant activity of BTE at various

concentrations against DPPH scavenging activity.

*The data was presented as mean ± SD. The assay was

done in triplicate for each concentration. The different

letter (a.b.c.d) showed significant difference among

BTE concentration (P<0.05).

Research by Leslie & Gunawan (2019), the

DPPH scavenging activity of BTE has IC50 137.60

µg/ml. The difference in the value of IC50 means

that the BTE has the differences between each

research. The differences included the genotype, the

method of extraction, the source of black tea, etc.

The DPPH scavenging activity of BTE was lower

than previous research (0.48 µg/ml) because, in this

research using filler mannitol, lactose, and starch 10-

50% which added in extract, it will lower the active

compound of BTE and decrease the anti-oxidant

activity.

Potential of Black Tea (Camellia Sinensis (L.) O. Kuntze) Extract as Anti-oxidant and Skin Anti-aging

3.5 ABTS Reducing Activity

BTE has anti-oxidant activity in reducing ABTS

with higher concentration BTE increase the ABTS

reducing activity (Figure 3). The IC50 value ABTS

reducing the activity of BTE was 17.21 μg/ml, it was

categorized highly active anti-oxidant (Marjoni and

Zulfisa, 2017).

Figure 3: Anti-oxidant activity of BTE at various

concentrations against ABTS scavenging activity.

*The data was presented as mean ± SD. The assay was

done in triplicate for each concentration. The different

letter (a.b.c.d.e) showed significant difference among BTE

concentration (P<0.05).

The IC50 value of ABTS scavenging activity of

BTE was 17.68 µg/ml which means with 15.29

µg/ml, it was categorized as a very strong activity

(Marjoni and Zulfisa, 2017). This result was similar

to previous research that BTE had active anti-

oxidant because it contained high secondary

metabolites namely epigallocatechin gallate

(EGCG)> epigallocatechin (EGC)>epicatechin (EC)

= catechin. EGCG is the most effective anti-oxidant

polyphenol against free radicals (He et al., 2018).

C. sinensis tea contains high polyphenol (30%) with

EGCG consists of 9% of that total polyphenol

(Crozier et al., 2012).

3.6 Skin Anti-aging Activity

The result of the skin anti-aging activity is shown in

Figure 4 and Table 1. Based on the data (Figure 4,

Table 1) showed that a higher concentration of BTE

increased inhibition activity toward elastase and

collagenase inhibition. Based on the result, the IC50

value of collagenase inhibition activity of BTE was

categorized as less active in skin anti-aging activity

when the IC50 value > 100.00 µg/ml (Vijayakumar

et al., 2017)., and BTE has categorized moderate

active elastase inhibition activity (IC50: 15.01-50.00

µg/ml) (Vijayakumar et al., 2017).

Figure 4: Skin anti-aging activity of BTE at various

concentrations against collagenase and elastase inhibition

activity.

*The data was presented as mean ± SD. The assay was

done in triplicate for each concentration. The different

letter (a.ab,bc.d.e) for collagenase and elastase inhibition

showed significant difference among BTE concentration

(P<0.05).

Table 1: The value of IC50 for collagenase and elastase

inhibition activity of BTE.

Sample

of Collagenase

Inhibition Activity

(µg/ml)

of Elastase

Inhibition Activity

(µg/ml)

BTE 123.72 ± 2.44 31.34 ± 1.70

*The data was presented as mean ±SD. The IC

value

was

calculated according to regression linear y=a+bx

BTE has collagenase and elastase inhibition

activity with IC50 values is 123.72 µg/ml and 31.34

µg/ml, respectively. From the IC50 value, BTE has

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

higher elastase inhibition activity than collagenase.

The anti-aging properties of black tea are because of

the presence of high polyphenol content. The

polyphenol that is responsible for anti-aging

properties is catechin (Khanna & Maurya, 2012).

The hydroxyl groups of polyphenol and flavonoid

chemicals are effective at forming bonds with the

carboxyl groups of the serine amino acid at the

elastase enzyme's active site, altering the enzyme's

mechanism of action. As a result, elastase is unable

to cleave peptide bonds, which considerably aids in

the prevention of skin elasticity loss and wrinkle

formation (Vijayakumar et al., 2017).

Collagenase is a zinc-containing

metalloproteinase, catechins are known as metal

chelators that may bind to the Zn2+ ion within

collagenase that prevent it from binding with the

substrate (Zeng et al., 2019; Voos et al., 2021).

Therefore, polyphenol content may bind to the Zn

ionactive site, preventing the substrate from

digesting the enzyme, and this mechanism could

contribute to the collagenase inhibition activity of

BTE (Pientaweeratch et al., 2016).

4 CONCLUSIONS

Black tea extract contains phenolic compound

52.81µg QE /mg sample and total flavonoid 10.96

µg QE /mg sample. The IC50 of black tea extract in

H2O2 scavenging activity 88.17±1.69 μg/ml, DPPH

scavenging activity 15.29±0.31 μg/ml and ABTS

17.21±1.22 μg/ml. Black tea extract has active to

highly active anti-oxidant properties. Anti-aging

activity namely collagenase inhibition with IC50

123.72±2.44 μg/ml as less activity and elastase

inhibition with IC50 31.34±1.70 μg/ml as moderate

active activity show that black tea extract has less

and moderately active in anti-aging properties.

ACKNOWLEDGEMENTS

This study was supported by the Grants-in-Aid from

Aretha Medika Utama Biomolecular and Biomedical

Research Center, Bandung, Indonesia. The author is

also thankful to Ervi Afifah, Muhammad Aldi

Maulana, Cintani Dewi Wahyuni, and Cahyaning

Riski Wijayanti from Biomolecular and Biomedical

Research Center, Aretha Medika Utama, Bandung,

Indonesia for their valuable assistance.

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